Gene transfer for studying and treating a connective tissue of a mammalian host

ABSTRACT

Methods for introducing at least one gene encoding a product into at least one target cell of a mammalian host for use in treating the mammalian host are disclosed. These methods include employing recombinant techniques to produce a vector molecule that contains the gene encoding for the product, and infecting the target cells of the mammalian host using the DNA vector molecule. A method to produce an animal model for the study of connective tissue pathology is also disclosed.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of and claims the benefit of U.S.application Ser. No. 08/924 777, filed Sep. 5, 1997, now U.S. Pat. No.6,156,304 the disclosure of which is incorporated by reference. This isa continuation-in-part application of U.S. application Ser. No.08/381,603, filed Jan. 27, 1995, now U.S. Pat. No. 5,858,355 acontinuation-in-part of U.S. application Ser. No. 08/685,212, filed Jul.23, 1996, now U.S. Pat. No. 6,228,356 which is a continuation of U.S.Ser. No. 08/027,750, filed Mar. 8, 1993, now abandoned, and acontinuation-in-part application of U.S. application Ser. No.08/567,710, filed Dec. 5, 1995, now abandoned which was a continuationof U.S. application Ser. No. 08/183,563, filed Jan. 18, 1994, nowabandoned, which was a continuation application of U.S. application Ser.No. 07/963,928, filed Oct. 20, 1992, now abandoned, which was acontinuation application of U.S. application Ser. No. 07/630,981, filedDec. 20, 1990, now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method of introducing at least onegene encoding a product into at least one cell of a mammalian host foruse in treating the mammalian host. This method discloses employingvector molecules containing a gene encoding the product and infecting,for example, connective tissue cell of the mammalian host using thevector molecule. This invention provides both viral and non-viralmethods of introducing at least one gene encoding a product into atleast one cell of the mammalian host to treat the mammalian host.

More specifically, the present invention discloses ex vivo and in vivotechniques for delivery of a DNA sequence of interest to the connectivetissue cells of the mammalian host. The ex vivo technique involves priorremoval and culture of target connective tissue cells, in vitroinfection of the DNA sequence by vector or other delivery vehicle intothe connective tissue cells, and transplantation of the modifiedconnective tissue cells to the mammalian host such as of a target joint,so as to effect in vivo expression of the gene product of interest. Asan alternative, non-connective tissue cells, such as hematopoieticprogenitor cells, stromal cells, bone marrow cells, myoblasts,leukocytes or mature lymphoid or myeloid cells may be transfected invitro, recovered and injected into the bone marrow or blood stream ofthe patient using techniques known to the skilled artisan. These cellscan also be injected locally into the connective tissue.

The in vivo technique bypasses the requirement for in vitro culture oftarget cells, instead relying on direct transplantation of the DNAsequence by vector or other delivery vehicle to the target cells invivo, thus effecting expression of the gene product of interest. Forexample, the gene encoding the product of interest can be introducedinto liposomes and injected directly into the area of the joint, wherethe liposomes fuse with synovial cells, resulting in an in vivo genetransfer to synovial tissue. Alternatively, the gene encoding theproduct of interest can be introduced into the area of the joint asnaked DNA. The naked DNA enters the synovial cell, resulting in an invivo gene transfer to synovial tissue.

The present invention also relates to a method for producing an animalmodel for the study of connective tissue pathologies and systemicindices of inflammation.

2. Brief Description of the Related Art

Arthritis involves inflammation of a joint that is usually accompaniedby pain and frequent changes in the structure of the joint. Arthritismay result from or be associated with a number of conditions includinginfection, immunological disturbances, trauma and degenerative jointdiseases, such as osteoarthritis. The biochemistry of cartilagedegradation in joints and cellular changes have received considerableinvestigation.

In a healthy joint, cells in cartilage (chondrocytes) and thesurrounding synovium (synoviocytes) are in a resting state. In thisresting state, these cells secrete basal levels of prostaglandins,cytokines and various proteinases, such as collagenase, gelatinase andstromelysin, with the ability to degrade cartilage. During thedevelopment of an arthritic condition, these cells become activated. Inthe activated state, synoviocytes and chondrocytes synthesize andsecrete large amounts of prostaglandins, cytokines and proteinases.

In efforts to identify pathophysiologically relevant cell activators, ithas been known that the cytokine interleukin-1 activates chondrocytesand synoviocytes and induces cartilage breakdown in vitro and in vivo.Additionally, interleukin-1 is a growth factor for synoviocytes andpromotes their synthesis of matrix, two properties suggesting theinvolvement of interleukin-1 in the synovial hypertrophy thataccompanies arthritis. In contrast, interleukin-1 inhibits cartilaginousmatrix synthesis by chondrocytes, thereby suppressing repair ofcartilage. Interleukin-1 also induces bone resorption and thus mayaccount for the loss of bone density seen in rheumatoid arthritis.Interleukin-1 is inflammatory, serves as a growth factor forlymphocytes, is a chemotactic factor and a possible activator ofpolymorphonuclear leukocytes (PMNs). When present in a sufficientconcentration, interleukin-1 may cause fever, muscle wasting andsleepiness.

The major source of interleukin-1 in the joint is the synovium.Interleukin-1 is secreted by the resident synoviocytes, which are joinedunder inflammatory conditions by macrophages and other white bloodcells.

Much attention has been devoted to the development of a class of agentsidentified as the “Non-Steroidal Anti-Inflammatory Drugs” (hereinafter“NSAIDs”). The NSAIDs inhibit cartilage synthesis and repair and controlinflammation. The mechanism of action of the NSAIDs appears to beassociated principally with the inhibition of prostaglandin synthesis inbody tissues. Most of this development has involved the synthesis ofbetter inhibitors of cyclo-oxygenase, a key enzyme that catalyzes theformation of prostaglandin precursors (endoperoxides) from arachidonicacid. The anti-inflammatory effect of the NSAIDs is thought to be due inpart to inhibition of prostaglandin synthesis and release duringinflammation. Prostaglandins are also believed to play a role inmodulating the rate and extent of leukocyte infiltration duringinflammation. The NSAIDs include drugs such as acetylsalicylic acid(aspirin), fenoprofen calcium (Nalfon® Pulvules®, Dista ProductsCompany), ibuprofen (Motrin®, The Upjohn Company), and indomethacin(Indocin®, Merck and Company, Inc.).

Therapeutic intervention in arthritis is hindered by the inability totarget drugs, such as the NSAIDs, to specific areas within a mammalianhost, such as a joint. Traditional routes of drug delivery, such asoral, intravenous or intramuscular administration, depend upon vascularperfusion of the synovium to carry the drug to the joint. This isinefficient because transynovial transfer of small molecules from thesynovial capillaries to the joint space occurs generally by passivediffusion. This diffusion is less efficient with increased size of thetarget molecule. Thus, the access of large drug molecules, for example,proteins, to the joint space is substantially restricted.Intra-articular injection of drugs circumvents those limitations;however, the half-life of drugs administered intraarticularly isgenerally short. Another disadvantage of intra-articular injection ofdrugs is that frequent repeated injections are necessary to obtainacceptable drug levels at the joint spaces for treating a chroniccondition, such as arthritis. Because therapeutic agents heretoforecould not be selectively targeted to joints, it was necessary to exposethe mammalian host to systemically high concentrations of drugs in orderto achieve a sustained, intra-articular therapeutic dose. Exposure ofnon-target organs in this manner exacerbated the tendency ofanti-arthritis drugs to produce serious side effects, such asgastrointestinal upset and changes in the hematological, cardiovascular,hepatic and renal systems of the mammalian host.

It has been shown that genetic material can be introduced into mammaliancells by chemical or biological means. Moreover, the introduced geneticmaterial can be expressed so that high levels of a specific protein canbe synthesized by the host cell. Cells retaining the introduced geneticmaterial may include an antibiotic resistance gene thus providing aselectable marker for preferential growth of the transduced cell in thepresence of the corresponding antibiotic. Chemical compounds forinhibiting the production of interleukin-1 are also known.

U.S. Pat. No. 4,778,806 discloses a method of inhibiting the productionof interleukin-1 by monocytes and/or macrophages in a human byadministering through the parenteral route a2-2′-[1,3-propan-2-onediyl-bis (thio)] bis-1 H-imidazole or apharmaceutically acceptable salt thereof. This patent discloses achemical compound for inhibiting the production of interleukin-1. Bycontrast, in one embodiment of the present invention, gene therapy isemployed that is capable of binding to and neutralizing interleukin-1.

U.S. Pat. No. 4,780,470 discloses a method of inhibiting the productionof interleukin-1 by monocytes in a human by administering a 4,5-diaryl-2(substituted) imidazole. This patent also discloses a chemical compoundfor inhibiting the production of interleukin-1.

U.S. Pat. No. 4,794,114 discloses a method of inhibiting the5-lipoxygenase pathway in a human by administering a diaryl-substitutedimidazole fused to a thiazole, pyrrolidine or piperidine ring or apharmaceutically acceptable salt thereof. This patent also discloses achemical compound for inhibiting the production of interleukin-1.

U.S. Pat. No. 4,870,101 discloses a method for inhibiting the release ofinterleukin-1 and for alleviating interleukin-1 mediated conditions byadministering an effective amount of a pharmaceutically acceptableanti-oxidant compound such as disulfiram, tetrakis[3-(2,6-di-tert-butyl-4-hydroxyphenyl) propionyloxy methyl] methane or2,4-di-isobutyl-6-(N,N-dimethylamino methyl)-phenol. This patentdiscloses a chemical compound for inhibiting the release ofinterleukin-1.

U.S. Pat. No. 4,816,436 discloses a process for the use of interleukin-1as an anti-arthritic agent. This patent states that interleukin-1, inassociation with a pharmaceutical carrier, may be administered byintra-articular injection for the treatment of arthritis orinflammation. In contrast, the present invention discloses a method ofusing and preparing a gene that is capable of binding to andneutralizing interleukin-1 as a method of resisting arthritis.

U.S. Pat. No. 4,935,343 discloses an immunoassay method for thedetection of interleukin-1 beta that employs a monoclonal antibody thatbinds to interleukin-1 beta but does not bind to interleukin-1 alpha.This patent discloses that the monoclonal antibody binds tointerleukin-1 beta and blocks the binding of interleukin-1 beta tointerleukin-1 receptors, and thus blocking the biological activity ofinterleukin-1 beta. The monoclonal antibody disclosed in this patent maybe obtained by production of an immunogen through genetic engineeringusing recombinant DNA technology. The immunogen is injected into a mouseand thereafter spleen cells of the mouse are immortalized by fusing thespleen cells with myeloma cells. The resulting cells include the hybridcontinuous cell lines (hybridomas) that may be later screened formonoclonal antibodies. This patent states that the monoclonal antibodiesof the invention may be used therapeutically, such as for example, inthe immunization of a patient, or the monoclonal antibodies may be boundto a toxin to form an immunotoxin or to a radioactive material or drugto form a radio pharmaceutical or pharmaceutical.

U.S. Pat. No. 4,766,069 discloses a recombinant DNA cloning vehiclehaving a DNA sequence comprising the human interleukin-1 gene DNAsequence. This patent provides a process for preparing humaninterleukin-1 beta, and recovering the human interleukin-1 beta. Thispatent discloses use of interleukin-1 as an immunological reagent inhumans because of its ability to stimulate T-cells and B-cells andincrease immunoglobulin synthesis.

U.S. Pat. No. 4,396,601 discloses a method for providing mammalian hostswith additional genetic capability. This patent provides that host cellscapable of regeneration are removed from the host and treated withgenetic material including at least one marker which allows forselective advantage for the host cells in which the genetic material iscapable of expression and replication. This patent states that themodified host cells are then returned to the host under regenerativeconditions.

U.S. Pat. No. 4,968,607 discloses a DNA sequence encoding a mammalianinterleukin-1 receptor protein which exhibits interleukin-1 bindingactivity.

U.S. Pat. No. 5,081,228 discloses a DNA sequence encoding both themurine and human interleukin-1 receptor. This patent also provides aprocess for the in vitro expression of said DNA sequences.

U.S. Pat. No. 5,180,812 discloses a substantially pure preparation ofthe human interleukin-1 receptor protein.

Patent Application WO9634955 discloses a method of treating an arthriticcondition using recombinantly modified articular chondrocytes.

U.S. Pat. No. 5,643,752 discloses a host cell transformed with anexpression vector containing nucleic acid amino acids 30-224 of theTIMP-4 polypeptide.

Patent Application WO9723639 discloses expression vectors containing DNAencoding a protein having the formula A-X-B, where A and B are subunitsof a dimeric protein or are each a biologically active protein; X is alinker polypeptide. Transformed hosts containing the vectors are alsodisclosed. The method reportedly can be used for the production ofinterleukin-12 using, DNA coding for the 40 Kd and 35 Kd subunits ofIL-12, joined by a suitable linker.

Patent Application WO9700958 discloses an isolated nucleic acid encodingpCL13, a member of TGF-β family member, having immunosuppressant, celldifferentiation promoting and anti-proliferative activities.

In spite of these disclosures, there remains a very real and substantialneed for a method for introducing at least one gene encoding a productof interest into at least one cell of a mammalian host in vitro, oralternatively in vivo, for use in treating the mammalian host. Further,there is a need for a process wherein a gene encoding a solubleinterleukin-1 receptor is used to resist the deleterious pathologicalchanges associated with arthritis. There is also a need to utilize oneor more DNA sequences for delivery to and expression of a protein orprotein fragment within a target host connective tissue cell, such as asynovial cell, or non-connective tissue cell so as to effect a treatmentof various joint pathologies and concomitant systemic indices ofinflammation. A further need exists to provide an animal model for thestudy of joint pathologies.

SUMMARY OF THE INVENTION

The present invention has met the above needs. A method of introducingat least one gene encoding a product of interest into at least one cellof a mammalian host for use in treating the mammalian host is providedby the present invention. This method includes employing recombinanttechniques to produce a vector molecule containing the gene encoding forthe product and infecting the target cell of the mammalian host usingthe vector molecule containing the gene. The vector molecule can be anymolecule capable of being delivered and maintained within the targetcell or tissue such that the gene encoding the product of interest canbe stably expressed. The vector molecule preferably utilized in thepresent invention is either a viral or retroviral vector molecule or aplasmid DNA non-viral molecule. This method preferably includesintroducing the gene encoding the product into the cell of the mammalianconnective tissue for a therapeutic or prophylactic use. Unlike previouspharmacological efforts, the methods of the present invention employgene therapy to address the chronic debilitating effects of jointpathologies.

One ex vivo method of treating a connective tissue disorder disclosedthroughout this specification comprises generating a recombinant viralvector which contains at least one DNA sequence encoding a protein orbiologically active fragment thereof. This recombinant viral vector isthen used to infect a population of in vitro cultured connective tissuecells or non-connective tissue cells, resulting in a population oftransduced cells. These transduced cells are then transplanted to thehost, for example in a target joint space, bone marrow, or blood streamof a mammalian host, effecting subsequent expression of the protein orprotein fragment within the joint space of the host. Expression of theDNA sequence of interest is useful in substantially reducing at leastone deleterious joint pathology or indicia of inflammation normallyassociated with a connective tissue disorder.

The methods of the present invention include employing a gene thatencodes one or more of the materials selected from the group consistingof (a) a human interleukin-1 receptor antagonist protein (IRAP); (b) aLac Z marker gene capable of encoding a beta-galactosidase protein; (c)a soluble interleukin-1 receptor protein (sIL-1R); (d) a soluble TNF-αreceptor protein (sTNF-αR); (e) a proteinase inhibitor; (f) a cytokine;(g) CTLA4;(h) FasL; (i) BMP; (j) an anti-adhesion molecule; (k) a freeradical antagonist; and (l) iNOS. Biologically active derivatives andfragments of these proteins are also within the scope of the presentinvention.

The genes used in the present methods can be introduced to the host invarious ways. One embodiment of the invention employs a viral vector.Preferably the viral vector is a vector selected from the group whichincludes (a) a retroviral vector such as MFG or pLJ, (b) anadeno-associated virus, (c) an adenovirus, and (d) a herpes virus,including but not limited to herpes simplex 1 or herpes simplex 2.

Another ex vivo embodiment if the invention employs a DNA plasmidvector. The DNA plasmid vector can be any DNA plasmid vector known toone of ordinary skill in the art capable of stable maintenance withinthe targeted cell or tissue upon delivery, regardless of the method ofdelivery utilized. One such method is the direct delivery of the DNAvector molecule, whether it be a viral or plasmid DNA vector molecule,to the target cell or tissue.

Another ex vivo embodiment of this invention employs non-viral means forintroducing the gene encoding for the product into the target cell ofthe host. More specifically, this method includes employing non-viralmeans selected from the group which includes (a) at least one liposome,(b) Ca₃(PO₄)₂, (c) electroporation, (d) DEAE-dextran, and (e) injectionof naked DNA. Transfected cells are then introduced to the host.

A further ex vivo embodiment of this invention includes employing thebiological means of utilizing a virus to deliver the DNA vector moleculeto the target cell or tissue. Preferably, the virus is a pseudo-typeretrovirus, the genome having been altered such that the pseudo-typeretrovirus is capable only of delivery and stable maintenance within thetarget cell, but not retaining an ability to replicate within the targetcell or tissue. The altered viral genome is further manipulated byrecombinant DNA techniques such that the viral genome acts as a DNAvector molecule containing the gene of interest to be expressed withinthe target cell or tissue.

In vivo methods can also be used to introduce the gene of interest tothe host cell. Viral or non-viral vectors can all be used for in vivotransduction or transfection of host cells, as can non-viral andbiological means as described above. The step of in vitro transductionor transfection by these vehicles is eliminated in the in vivo methods,as the vehicle containing the DNA sequence is introduced directly to thehost. Infection with the gene(s) of interest occurs in vivo.

A further embodiment of this invention provides for an animal model andmethod of producing an animal model to study connective tissuepathologies and indices of systemic inflammation. This model utilizeseither ex vivo or in vivo delivery of at least one gene or DNA sequenceof interest into at least one cell of a mammalian host. Examples ofjoint pathologies which can be studied in the present invention include,but are not limited to, joint pathologies such as leukocytosis,synovitis, cartilage breakdown and suppression of cartilage matrixsynchesis. Examples of indices of systemic inflammation include, but arenot limited to, elevated erythrocyte sedimentation rate, fever andweight loss.

More specifically, the animal model embodiments for the study of jointpathologies generally comprise generating a recombinant viral or plasmidvector which contains a DNA sequence encoding a protein, or biologicallyactive derivative or fragment thereof, known to cause or contribute toone or more of the joint pathologies or symptoms associated with aconnective tissue disorder; infecting a population of in vitro culturedtarget cells with said recombinant viral vector, resulting in apopulation of transfected target cells; and transplanting saidtransfected connective cells to a joint space of a mammalian host. TheDNA sequence used in the animal model embodiments encode a compoundknown to be associated with the deleterious effects of a connectivetissue disorder. DNA sequences particularly suited for animal modelstudies are DNA sequences encoding interleukin-1α (IL-1α), IL-1β, IL-2,IL-8, IL-12, IL-15, IL-17, tumor necrosis factor α (TNF-α), TNF-β andproteinases such as gelatinase, stromelysin, collagenase and aggrecanaseor biologically active derivatives or fragments thereof. Subsequentexpression of these proteins within the joint space of the hosttherefore causes at least one deleterious joint pathology or indicia ofinflammation normally associated with a connective tissue disorder inthe afflicted joint. The animal in which arthritis is induced can thenbe used to test the efficacy of various methods devised to reduce thesymptoms associated with a connective tissue disorder.

Means for delivering genes of interest include, for example, therecombinant viral vectors, plasmid vectors, non-viral means orbiological means described above in connection with the therapeuticmethods of the present invention. In vivo methods as described above canalso be used to introduce the DNA sequence of interest directly to theanimal host joint.

In a specific method disclosed as an example, and not as a limitation tothe present invention, a DNA plasmid vector containing the interleukin-1beta (IL-1β) coding sequence was ligated downstream of thecytomegalovirus (CMV) promoter. This DNA plasmid construction wasencapsulated within liposomes and injected intra-articularly into theknee joints of recipient rabbits. IL-1β was expressed and significantamounts of IL-1β were recovered from the synovial tissue. An alternativeis injection of the naked plasmid DNA into the knee joint, allowingdirect transfection of the DNA into the synovial tissue. Injection ofIL-1β into the joint of a mammalian host allows for prolonged study ofvarious joint pathologies and systemic indices of inflammation, asdescribed within this specification.

Another example demonstrates the introduction and subsequent expressionof human interleukin-1β (hIL-1β) into rabbit knee joints using theretroviral vector DFG-hIL-1-neo. Again, this resulted in a severeinflammatory arthritis, having pathophysiological changes consistentwith those seen in human arthritic conditions. Thus the presentinvention provides a reliable animal model for the study of connectivetissue disorders, which is predictive of human studies. An animal modelas described and exemplified in this specification measures the abilityof various gene therapy applications disclosed throughout thisspecification to withstand challenges from known causative agents (suchas IL-1β) of joint pathologies and inflammatory side effects.

A preferred method of the present invention for the therapeutictreatment of a host involves delivering the IRAP gene to the synoviallining of a mammalian host through use of a retroviral vector with theex vivo technique disclosed within this specification. A DNA sequence ofinterest encoding a functional IRAP protein or protein fragment issubcloned into a retroviral vector of choice, the recombinant viralvector is then grown to adequate titers and used to infect in vitrocultured synovial cells, and the transduced synovial cells, preferablyautografted cells, are transplanted into the joint of interest,preferably by intra-articular injection. Other preferred methods of thepresent invention involve delivery of vIL-10, IL-10, sTNF-αR, and sIL-1Rinstead of IRAP.

Another preferred method of the present invention involves direct invivo delivery of the IRAP gene to the synovial lining of a mammalianhost through use of either an adenovirus vector, an adeno-associatedvirus (AAV) vector or a herpes-simplex virus (HSV) vector. A DNAsequence of interest encoding a functional IRAP protein or proteinfragment is subcloned into the respective viral vector, theIRAP-containing viral vector is then grown to adequate titers, anddirected into the joint space, preferably by intra-articular injection.A retroviral-IRAP construct, such as MFG-IRAP may also be utilized todirectly target cells within the joint space. Preferred variations ofthis method for in vivo delivery can be performed using DNA sequencesencoding sTNF-αR, sIL-1R, one or more of the cytokines that possessanti-inflammatory and/or immunomodulatory characteristics such as IL-4,IL-10 or IL-13, anti-adhesion molecules that inhibit cell-cell andcell-matrix interactions including but not limited to soluble ICAM-1 andsoluble CD44, cartilage growth factors including but not limited toIGF-1 and TGF-β, free radical antagonists that reduce the deleteriouseffects of free radical formation within the joint including, but notlimited to, superoxide dismutase and proteins or protein fragments thatinhibit NO and NO synthase. Biologically active derivatives andfragments of the proteins encoded by these DNA sequences are also withinthe scope of the present invention.

Direct intraarticular injection of a DNA molecule containing the gene ofinterest into the joint results in transfection of the recipient targetcells and hence bypasses the requirement of removal, in vitro culturing,transfection, selection, and transplantation of the DNA vectorcontaining cells to promote stable expression of the heterologous geneof interest. Methods of presenting the DNA molecule to the target cellsof the joint include, but are not limited to, association of the DNAmolecule with cationic liposomes, subcloning the DNA sequence ofinterest in a retroviral vector as described throughout thisspecification, or the direct injection of the DNA molecule itself intothe joint. The DNA molecule, regardless of the form of presentation tothe knee joint, is preferably presented as a vector molecule, either asa recombinant viral DNA vector molecule or a recombinant DNA plasmidvector molecule. Expression of the heterologous gene of interest isensured by inserting a promoter fragment active in eukaryotic cellsdirectly upstream of the coding region of the heterologous gene. One ofordinary skill in the art may utilize known strategies and techniques ofvector construction to ensure appropriate levels of expressionsubsequent to entry of the DNA molecule into the target cell or tissue.In vivo delivery of various viral and non-viral vectors to the rabbitknee joint are described in the Examples.

A preferred method of using the gene coding for the solubleinterleukin-1 receptor (sIL-1R) of this invention involves employingrecombinant techniques to generate a cell line which produces infectiousviral particles containing the gene coding for sIL-1R. The producer cellline is generated by inserting the gene into a viral vector under theregulation of a suitable eukaryotic promoter, transfecting the viralvector containing the gene into the viral packaging cell line for theproduction of a viral particle capable of expressing the gene coding forsIL-1R, and infecting synovial or other cells of a mammalian host usingthe viral particle. The cells can be infected in culture (ex vivo) withviral particles and subsequently transplanted back into the joint, orcan be infected in vivo by direct administration of the viral particlesto the host joint. This method may be employed in both prophylactic andtherapeutic treatment of joint pathologies in any joint area.

More specifically, a preferred method of using the gene coding for thesIL-1R involves introducing the viral particles obtained from theretroviral packaging cell line directly by intra-articular injectioninto a joint space of a mammalian host that is lined with synovialcells. In a preferred embodiment, synoviocytes recovered from the kneejoint are cultured in vitro for subsequent utilization as a deliverysystem for gene therapy. It will be apparent that tissue other thansynovial can be used. It would be possible to utilize other connectivetissue sources, such as skin cells, or non-connective tissue cells, forin vitro culture techniques. The method of using the gene of thisinvention may be employed both prophylactically and in the therapeutictreatment of arthritis in any susceptible joint.

Another embodiment of this invention provides a method for preparing agene encoding a product of interest including synthesizing the gene by apolymerase chain reaction, introducing the amplified coding sequenceinto a retroviral vector, transfecting the retroviral vector into aretrovirus packaging cell line and collecting viral particles from theretrovirus packaging cell line. A compound for parenteral administrationto a patient in a therapeutically or prophylactically effective amountcontaining a gene encoding sIL-1R in a suitable pharmaceutical carrieris also provided for in the present invention. Such compounds can alsobe prepared using one or more of the other genes of interest disclosedherein.

The methods of the present invention involve transfection ortransduction of numerous types of cells including connective tissuecells such as ligaments, cartilage, tendon, synovium, skin, bone,meniscus and intervertebral disc tissue, and non-connective tissue cellssuch as hematopoietic progenitor cells, stromal cells, bone marrowcells, myoblasts, leukocytes or mature lymphoid or myeloid cells, with avector molecule containing any of the gene or genes disclosed throughoutthe specification. The transfected cells are recovered and injected intothe host, such as in the joint space, bone marrow or blood stream, usingtechniques known and available to one of ordinary skill in the art. Itwill be possible, within the scope of this method, to use cells derivedfrom autologous bone marrow instead of cells derived from donor bonemarrow so as to modify rejection.

It is an object of the present invention to provide a method ofintroducing at least one gene encoding a product into at least onetarget cell of a mammalian host for use in treating the mammalian host.

It is an object of the invention to provide a method of introducing agene encoding a product into at least one target cell of a mammalianhost for a therapeutic use.

It is an object of the present invention to provide a method ofintroducing into the synovial lining cells of a mammalian arthriticjoint at least one gene that encodes a protein having therapeuticproperties.

It is an object of the present invention to provide an animal model forthe study of connective tissue pathology.

It is an object of the present invention to provide a method ofintroducing by ex vivo or in vivo methods a gene coding for the sIL-1Rthat is capable of binding to and neutralizing substantially allisoforms of interleukin-1, including interleukin-1 alpha andinterleukin-1 beta.

It is an object of the present invention to provide a method ofintroducing by ex vivo or in vivo methods a gene coding for IRAP or abiologically active derivative thereof, which is a competitive inhibitorof and therefore substantially neutralizes all isoforms ofinterleukin-1, including interleukin-1 alpha and interleukin-1 beta.

It is an object of the present invention to provide a method ofintroducing by ex vivo or in vivo methods a gene in a mammalian hostthat is capable of binding to and neutralizing substantially allisoforms of interleukin-1 and thus, substantially resist the degradationof cartilage and protect surrounding soft tissues of the joint space.

It is an object of the present invention to provide a method ofintroducing by ex vivo or in vivo methods a gene coding for the sIL-1Rthat is capable of binding to and neutralizing substantially allisoforms of interleukin-1 for the prevention of arthritis in patientsthat demonstrate a high susceptibility for developing the disease.

It is an object of the present invention to provide a method ofintroducing by ex vivo or in vivo methods a gene coding for IRAP that iscapable of acting as c Competitive inhibitor of and thereforesubstantially neutralizes all isoforms of interleukin-1 for theprevention of arthritis in patients that demonstrate a highsusceptibility for developing the disease.

It is an object of the present invention to provide a method ofintroducing by ex vivo or in vivo methods a gene coding for an sIL-1Rthat is capable of binding to and neutralizing substantially allisoforms of interleukin-1 for the treatment of patients with arthritis.

It is an object of the present invention to provide a method ofintroducing by ex vivo or in vivo methods a gene coding for IRAP or abiologically active derivative thereof, which is a competitive inhibitorof and therefore substantially neutralizes all isoforms of interleukin-1for the treatment of patients with arthritis.

It is an object of the present invention to provide a method ofintroducing by ex vivo or in vivo methods a gene or genes that addressthe chronic debilitating pathophysiology of arthritis.

It is a further object of the present invention to provide a compoundfor parenteral administration to a patient which comprises a geneencoding sIL-1R in a suitable pharmaceutical carrier.

It is a further object of the present invention to provide a compoundfor parenteral administration to a patient which comprises a geneencoding IRAP in a suitable pharmaceutical carrier.

These and other objects of the invention will be more fully understoodfrom the following description of the invention, the referenced drawingsand the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of the cDNA encoding the human interleukin-1receptor antagonist protein (IRAP) gene inserted into the NcoI and BamHIcloning sites of the retroviral vector MFG.

FIG. 2 shows the structure of the cDNA encoding the human interleukin-1receptor antagonist protein (IRAP) gene with a selectable neo markerinserted into the retroviral vector MFG.

FIG. 3 shows a micrograph of synovium recovered from the knee of arabbit approximately one month after intra-articular injection of LacZ⁺, neo⁺ synoviocytes employing the methods of this invention.

FIG. 4 shows a Western blot demonstrating the production ofinterleukin-1 receptor antagonist protein by four cultures of HIG-82cells (Georgescu, et al., 1988, In Vitro 24: 1015-1022) infected usingthe method of this invention employing the MFG-IRAP viral vector.

FIG. 5 shows data demonstrating the inhibition of gelatinase productionby chondrocytes by the addition of medium conditioned by MFG-IRAPinfected HIG-82 cells.

FIG. 6 shows the uptake and expression of the Lac Z gene by synoviocytesusing lipofection. Well 1—Control cells, treated with liposomes alone;Well 2—Control cells, treated with DNA alone; Well 3—DNA+150 nmoleliposomes; Well 4—DNA+240 nmole liposomes; Well 5—DNA+300 nmoleliposomes; Well 6—DNA+600 nmole liposomes.

FIG. 7 shows the interleukin-1 binding domain amino acid arrangement.

FIGS. 8A-8C show the amino acid and nucleotide sequence of the human(SEQ ID NOS 1 and 2) and mouse (SEQ ID NOS 3 and 4) interleukin-1receptors.

FIG. 9 shows gene encoding a soluble interleukin-1 receptor insertedinto a retroviral vector.

FIG. 10 shows anti-inflammatory properties of the MFG-IRAP transgene.MFG-IRAP/HIG-82 cells (10⁷) or untransduced HIG-82 cells (10⁷) weretransplanted to the knee joints of rabbits 3 days before intraarticularchallenge with the indicated amounts of recombinant human interleukin-1beta (rhIL-1β). Lavage of joints occurred 18 hours later, after whichinfiltrating leukocytes were counted.

FIG. 11 shows levels of human IRAP in rabbit knees four days followingtransplant of synoviocytes. Either untransduced (naive) HIG-82 cells orcells carrying a human IRAP gene (MGF-IRAP/HIG-82) were injectedintraarticularly in the knee joints or rabbits (10⁷ cells/knee). Fourdays later, knees were lavaged and the concentration of human IRAPdetermined by ELISA. Values given are means±S.D. (n=15).

FIGS. 12 A-C shows inhibition of IL-1 induced leukocyte infiltration inknees expressing IRAP gene. Either naive or IRAP-transduced HIG-82 cellswere transplanted into rabbit knee joints, as indicated Three days later0-100 pg/knee hIL-1β was intraarticularly injected at the indicateddoses. The following day, knee joints were lavaged and the leukocyticinfiltrate analyzed by counting with a hemocytometer and bycytospinning. Means±S.E. (n=3). (A) White blood cells (WBC) per knee.(B) Stained cytospin preparation of lavages from control knee injectedwith IL-1. Preparation was diluted 1:10 prior to cytospinning. (C)Stained cytospin preparation of lavages from IRAP-secreting kneeinjected with IL-1. The preparation was not diluted.

FIG. 13 shows suppression of IL-1 induced loss of proteoglycans fromarticular cartilage. Either naive or IRAP-transduced HIG-82 cells weretransplanted into rabbits knee joints. Three days later, 0-200 pg/kneehrIL-1 was intraarticularly injected at the indicated doses. Thefollowing day, knee joints were lavaged and the level ofglycosaminoglycans (GAG) as an index of cartilage breakdown wasdetermined.

FIGS. 14A-D shows suppression of IL-1 mediated synovial changes in kneesexpressing IRAP. Ten pg hrIL-1B was injected intraarticularly in eachcase. Synovia were harvested 18 hours after injection of IL-1β, i.e. 4days after transplantation of naive or IRAP-secreting HIG-82 cells. (A)Control synovium following injection of IL-1, magnification×10. (B)IRAP-secreting synovium following injection of IL-1, magnification×10.(C) Control synovium following injection of IL-1, magnification×160. (D)IRAP-secreting synovium magnification×160.

FIG. 15 shows expression of human IRAP in normal and arthritic knees ofrabbits. Antigen-induced arthritis was initiated by injecting 5 mgovalbumin into one knee joint (arthritic knee) of pre-sensitized rabbitson day 1. The contralateral knee (non-arthritic knee) received carriersolution only. On day 2, autologous synoviocytes (10⁷/knee in 1 mlsaline) were transferred to selected knee joints by intraarticularinjection. Certain non-arthritic knees and arthritic knees receivedcells transduced with the human IRAP gene. Other non-arthritic andarthritic knees received untransduced cells or cells transduced with lacZ and neo^(r) genes (controls). As the results obtained with these twotypes of control cells were indistinguishable, they have been pooled inthe figures. Detailed methods for synoviocyte culture, transduction andintraarticular implantation are disclosed throughout this specification.

On day 4, knees were lavaged with 1 ml saline. On day 7, rabbits werekilled and the knees again lavaged. The concentrations of human IRAP inthe lavage fluids were determined by ELISA using a commercial kit (R&DSystems, Minneapolis, Minn.). Values given are means±S.E. Numbers ofknees are shown above each column. Asterisks denote values which differat p<0.05 (t-test).

FIG. 16 shows concentrations of rabbit IL-1β in the normal and arthriticknee joints of rabbits. Experimental conditions were identical to thosedescribed in FIG. 15. However, lavage fluids were assayed for rabbitIL-1α and rabbit IL-1β by RIA using a commercial kit (Cytokine Sciences,Boston, Mass.). Low levels of IL-1β are present in non-arthritic kneesas a reflection of the slight inflammatory effects provoked byintraarticular injection. No IL-1α was detectable in any of the samples.Values given are means±S.E. Numbers of knees are shown above eachcolumn. Asterisks denote values which differ at p<0.05 (t-test).

FIGS. 17A-B shows the effect of IRAP gene transfer on cartilage matrixmetabolism. Experimental conditions were as described for FIG. 15,except that rabbits were killed both at days 4 and 7. GAG concentrationsin the lavage fluids (FIG. 17A) were measured spectrophotometrically bythe dimethymethylene blue assay (Farndale, et al., Biochim. Biophys.Acta. 883: 173-177 (1986)). Fragments of articular cartilage were shavedfrom the femoral condyles of the knees and GAG synthesis (FIG. 17B) wasmeasured as the uptake of ³⁵SO₄ ²⁻ into macromolecular material asdescribed (Taskiran, et al., Biochem. Biophys. Res. Commun. 200:142-148(1994)). Results are shown in each case as percent of control valuesgiven are means±S.E. Numbers of knees are shown above each column.

FIG. 18 shows effects of IRAP gene transfer on leukocytosis.Experimental conditions were identical to those described in FIG. 15.Numbers of leukocytes in the lavage fluids were determined with ahemocytometer. Values shown are means±S.E. Numbers of knees are shownabove each column. Asterisks denote values which differ at p<0.05(t-test).

FIGS. 19A-D shows intraarticular expression of hIL-1β and its pathogeniceffects determined according to the methods of Example XVI.

FIG. 20 shows levels of human (h), rabbit (r) IL-1β and rabbit (r) TNF-αrecovered in lavage fluids determined according to the methods ofExample XVI. All values are expressed as the mean±S.E.M.

FIGS. 21A-D shows detection of hIL-1β expression in vivo and its grossto pathology determined according to the methods of Example XVI.

FIGS. 22A-F shows local and systemic effects following intraarticulartransplantation of autologous hIL-1β+synoviocytes determined accordingto the methods of Example XVI. 2.5×10⁶ naive synoviocytes (Control) orhIL-1β+synoviocytes (hIL-1) were autografted into the right and leftknees, respectively, of twelve rabbits at day 0. Three rabbits weresacrificed at day 7, 4 at day 14, and 5 at day 28. For A, D, E and Feach time point reflects measurements taken on remaining rabbits priorto sacrifice; B, and C reflect results obtained from rabbits sacrificedat that time point.

FIGS. 23A-H shows joint histology following expression of hIL-1βdetermined according to the methods of Example XVI. FIGS. 23 (A)-(F) aresynovial sections stained with hematoxylin and eosin; (G) and (H) aresections of femoral condyles stained with toluidine blue.

FIG. 24 upper panel shows the intraarticular expression levels of mIL-6delivered by ex vivo gene transfer and FIG. 24 lower panel shows theeffect of mIL-6 on GAG release, determined according to the methods ofExample XVIII.

FIG. 25 upper panel shows the intraarticular elevation in leukocyteinfiltration and FIG. 25 lower panel shows the depression of GAGsynthesis rates due to over expression of mTNF-α by ex vivo deliverydetermined according to the methods of Example XVIII.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “patient” includes members of the animalkingdom including but not limited to human beings.

As used herein, the term “mammalian host” includes mammalian members ofthe animal kingdom including but not limited to human beings.

As used herein, the term “target cells” refers to cells that aretargeted for transfection with the gene or genes encoding the product(s)of interest. Target cells can be either cells that are removed from ahost and cultured with the gene(s) in vitro and returned to a host in anex vivo methodology, or cells that are in the host and are transduced ortransfected in vivo. Generally a target cell when used in reference toex vivo methods is any cell that, when injected into a joint of apatient, will survive and express the gene. When used in reference to invivo methods, a target cell is any cell capable of being transduced ortransfected with one or more genes of interest and which willsubsequently express the gene. Target cells include both connectivetissue cells and non-connective tissue cells, as those terms are definedbelow.

As used herein, the term “connective tissue” includes but is not limitedto a ligament, a cartilage, a tendon, a synovium, skin, bone, meniscusand intervertebral disc tissue of a mammalian host.

As used herein, the term “non-connective tissue” includes but is notlimited to hematopoietic progenitor cells, stromal cells, bone marrowcells, leukocytes, and lymphoid or myeloid cells of a mammalian host.

As used herein, the terms “gene”, “DNA sequence” or “product” “ofinterest” refer to genes, DNA sequences or the products they encode thatare introduced to the host according to any of the methods of thepresent invention. For methods used in the therapeutic or prophylactictreatment of a host, the products of interest would be those proteins orpeptides, or fragments or derivatives thereof, that have therapeuticand/or prophylactic properties. For methods used in the animal model,the products of interest would be those proteins or peptides, orfragments or derivatives thereof, that have a pathologic effect on thehost, contributing to one or more of the deleterious effects ofconnective tissue disorders.

As used herein, the term “therapeutic” refers to the ability of a gene,product, protein, peptide, method and the like to alleviate at least onsymptom of a connective tissue disorder, or the benefit realized fromsuch alleviation. The term “prophylactic” refers to the ability of agene, product, protein, peptide, method and the like to prevent or atleast retard the onset of at least one symptom of a connective tissuedisorder, or the benefit realized from such action.

As used herein, the term “enhanced therapeutic benefit” refers to thetherapeutic benefit realized when more than one gene of interest isintroduced to a host at the same time; the enhanced therapeutic benefitis greater than the therapeutic benefit of each of the genesadministered separately. The benefit can be either additive orsynergistic.

As used herein, the term “DC-chol” means a cationic liposome containingcationic cholesterol derivatives. The “DC-chol” molecule includes atertiary amino group, a medium length spacer arm (two atoms) and acarbamoyl linker bond as described in Biochem. Biophys. Res. Commun.,179:280-285 (1991), X. Gao and L. Huang.

As used herein, “SF-chol” is defined as a type of cationic liposome.

As used herein, the term “biologically active” used in relation toliposomes denotes the ability to introduce functional DNA and/orproteins into the target cell.

As used herein, the term “biologically active” in reference to a nucleicacid, protein, protein fragment or derivative thereof is defined as anability of the nucleic acid or amino acid sequence to mimic a knownbiological function elicited by the wild type form of the nucleic acidor protein.

As will be appreciated by those skilled in the art, a fragment orderivative of a nucleic acid sequence or gene that encodes for a proteinor peptide can still function in the same manner as the entire wild typegene or sequence. Likewise, forms of nucleic and sequences can havevariations as compared with the wild type sequence, while the sequencestill encodes a protein or peptide, or fragments thereof, that retaintheir wild type function despite these variations. Similarly,derivatives of the genes and products of interest used in the presentinvention will have the same biological effect on the host as thenon-derivatized forms. Examples of such derivatives include but are notlimited to dimerized or oligomerized forms of the genes or proteins, aswells as the genes or proteins modified by the addition of animmunoglobulin (Ig) group. Proteins, protein fragments or derivativesthereof also can experience deviations from the wild type form whilestill functioning in the same manner as the wild type form. Biologicallyactive derivatives or fragments, when referring to the genes and/or DNAsequences described herein, are therefore also within the scope of thepresent invention.

One skilled in the art could test for the biological activity of aderivative or fragment of these genes/sequences/proteins by variousmethods known to those skilled in the art.

To determine if a fragment or derivative of IRAP is biologically active,a bioassay can be performed; if the compound blocks the ability ofinterleukin-1 to cause inflammation and cartilage breakdown, thederivative or fragment is a biologically active derivative or fragmentof IRAP. Similarly, a bioassay can be performed to determine if afragment or derivative of soluble interleukin-1 receptor protein isbiologically active by determining whether the compound blocks theability of interleukin-1 to cause inflammation and cartilage breakdown.To determine if a fragment or derivative of sTNF-αR is biologicallyactive, a bioassay can be performed; if the compound prevents cell deathin an L929 cell line in response to TNF-α, the fragment or derivative isbiologically active. To determine if a fragment or derivative of aproteinase inhibitor is biologically active, a bioassay can be performedto determine whether the action of a proteinase is inhibited, such as bymonitoring the rate of breakdown of a proteinaceous substrate.Inhibition of the proteinase would indicate biological activity. Forexample, the biological activity of a TIMP matrix metalloproteinaseinhibitor can be determined by its ability to inhibit the activity ofmatrix metalloproteinases, as assayed by methods described by Watanabeet al., Exp. Cell Res., 167:218-226 (1986). To determine if a fragmentor derivative of a therapeutic cytokine is biologically active, abioassay can be performed to determine if the cytokine has a therapeuticor prophylactic effect in inhibiting any of the symptoms associated witha connective tissue disorder. For example, the biological activity ofIL-6 can be determined by its ability to promote growth of B29 cells, asdescribed by Arden et al., Eur. J. Immunol., 17:1411-1416 (1987). Thebiological activity of IL-10 or vIL-10 can be determined by the abilityof derivatives or fragments of these compounds to inhibit the productionof nitric oxide by activated macrophages. To determine if a fragment orderivative of a growth hormone or a growth factor is biologicallyactive, bioassays can be performed as taught by Taskiran et al.,Biochem. Biophys. Res. Commun., 200:142-148 (1994); biologically activederivatives or fragments will demonstrate increased proteoglycansynthesis by cartilage. To determine if a fragment or derivative of ananti-adhesion molecule is biologically active, a bioassay can beperformed to determine the ability of the derivative or fragment toinhibit adhesion. To determine if a fragment or derivative of a freeradical antagonist is biologically active, a bioassay can be performedto determine the ability of the fragment or derivative to inhibit theproduction of free radicals. To determine if a derivative or fragment ofCTLA4 is biologically active, a bioassay can be performed to determineif the compound has the ability to bind to cells expressing B7.1, inwhich case it would be active. To determine if a derivative or fragmentof FasL is biologically active, a bioassay can be performed to determineif the compound has the ability to induce apoptosis of cells thatexpress Fas, in which case it would be biologically active. To determineif a derivative or fragment of iNOS is biologically active, a bioassaycan be performed to determine if the compound has the ability tosynthesize NO, in which case it would be biologically active. Any othermanner for determining biological activity known to those skilled in theart can also be used.

As used herein, the term “maintenance”, when used in the context ofliposome delivery, denotes the ability of the introduced DNA to remainpresent in the cell. When used in other contexts, it means the abilityof targeted DNA to remain present in the targeted cell or tissue so asto impart a therapeutic or prophylactic effect.

Connective tissues are difficult to target therapeutically. Intravenousand oral routes of drug delivery that are known in the art provide pooraccess to these connective tissues and have the disadvantage of exposingthe mammalian host body systemically to the therapeutic agent. Morespecifically, known intra-articular injection of joints provides directaccess to a joint. However, most of the injected drugs have a shortintraarticular half-life. The present invention solves these problems byintroducing into the mammalian host genes encoding for proteins that maybe used to treat the mammalian host. In a preferred embodiment, thisinvention provides a method for introducing into the connective tissueof a mammalian host genes encoding for proteins with anti-arthriticproperties.

The present invention provides a method for introducing at least onegene encoding a product into at least one target cell of a mammalianhost for use in treating the mammalian host, which comprises employingrecombinant techniques to generate a vector containing one or more DNAsequences encoding one or more products of interest, and infecting thecell of the mammalian host using the recombinant vector. This methodpreferably includes introducing the gene encoding the product into atleast one target cell of the mammalian host for a therapeutic orprophylactic use. Both in vivo and ex vivo methods can be used tointroduce the gene of interest to the host.

One ex vivo method for treating a connective tissue disorder accordingto the present invention comprises generating a recombinant vectorcontaining one or more DNA sequences encoding one or more genes ofinterest, or biologically active derivatives or fragments thereof;infecting a population of in vitro cultured target cells with thevector, resulting in a population of transduced target cells; andtransplanting the transduced cells to the mammalian host, effectingsubsequent expression of the protein or protein fragment within thehost. Expression of the protein or protein fragment of interest isuseful in reducing at least one deleterious joint pathology or indiciaof inflammation normally associated with a connective tissue disorder.Expression of the DNA sequence can also have a protective effect. Anymeans known to those skilled in the art can be used to introduce thetransduced target cells to the target joint space. Intraarticularinjection is preferred.

Any type of connective tissue cell or non-connective tissue cells, asthose terms are described herein, can be used. Preferably, if usingconnective tissue, synovial cells are used; more preferably, fortreating a human patient, the patient's own cells, such as autologoussynovial cells, are used. When ligament cells are used, preferably theligament is the medial collateral ligament (MCL). Use of cells and/ortissue from the patellar tendon and hamstring are also within the scopeof the invention. Preferably, if using non-connective tissue, stromalcells are used.

For the ex vivo methods, all of the non-connective tissue cells can beinjected back into the bone marrow or bloodstream of the host followingtransduction. Both connective and non-connective tissue cells can beinjected into the joint space, or any other area, of the host followingtransduction. For the in vivo methods, non-connective tissue cells canbe targeted in the bone marrow, bloodstream, joint space, or any otherarea, of the host and connective tissue cells can be targeted to anyarea of the host, preferably in the joint space.

Use of numerous genes, and biologically active derivatives and fragmentsthereof, are within the scope of the invention. Any gene capable ofmaintenance and expression, and encoding a product having a therapeuticand/or prophylactic effect in the treatment of joint pathology can beused in the methods of treating a host. These genes and biologicallyactive derivatives and fragments include, but are not limited to, DNAsequences encoding for one or more of: interleukin-1 receptor antagonistprotein (IRAP); a Lac Z marker gene capable of encoding abeta-galactosidase; a soluble interleukin-1 receptor (sIL-1R); a solubleTNF-α receptor (sTNF-αR); a proteinase inhibitor; a therapeuticcytokine; CTLA4; FasL; an anti-adhesion molecule; and a free radicalantagonist. Any other gene having therapeutic properties and DNA capableof maintenance and expression can also be used. These genes can beeither commercially obtained through any supplier or can be made by oneskilled in the art from cDNA libraries or through the reversetranscriptase polymerase chain reaction (RTPCR) method.

IRAP is a cytokine known to suppress the inflammatory responses causedby interleukin-1 in joint spaces. Introduction of IRAP to these spaces,therefore, causes a reduction in the inflammation associated with jointpathologies characterized as having IL-1 production. It is believed thatthe IRAP binds with the interleukin-1 receptors, thereby preventingbinding of the IL-1 to the receptors and inhibiting the inflammatoryeffects caused when IL-1 binds to the receptors, although the inventorsdo not wish to be bound by this mechanism.

Similarly, soluble interleukin-1 receptors (sIL-1R) bind to IL-1 withouttransmitting a cellular response, thereby preventing IL-1 from bindingto the native, cell surface receptors. Any sIL-1 receptor can be used,including but not limited to, Type I and Type II receptors; sIL-1R TypeII receptors are preferred because they do not bind to IRAP, while TypeI receptors do. The Type I sIL-1R is an 80 Kd glycoprotein that ispresent on T-lymphocytes, fibroblasts, and chondrocytes. The Type IIsIL-1R is 67 Kd in size and is found predominantly on macrophages andpre-B-cells.

Soluble tumor necrosis factor-alpha receptor (sTNF-αR) binds TNF-α andprevents it from having a damaging effect on the connective tissue of apatient. TNF-α is a cytokine which is known to contribute to thepathological effects of connective tissue disorders. The sTNF-αR of thepresent invention can be of any type, including Type I and Type II. TheType I sTNF-αR is an 55 Kd glycoprotein and Type II sTNFα-R is 75 Kd insize. Both receptors are widely distributed on various cell types. Boththe sIL-1R and sTNF-αR have been shown to alleviate at least some of thesymptoms associated with connective tissue disorders.

Various proteinase inhibitors are also within the scope of the presentinvention. Proteinase inhibitors are substances that prevent theenzymatic breakdown of proteins. Both proteinase inhibitors andmetalloproteinase inhibitors are within the scope of the invention;preferred proteinase inhibitors are tissue inhibitor ofmetalloproteinase (TIMP), TIMP-1, TIMP-2, TIMP-3 and TIMP-4, plasminogenactivator inhibitors (PAIs) and serpins.

Cytokines are small proteins with the properties of locally actinghormones. They serve to communicate between cells in a paracrine manner,and may also act in an autocrine manner on the same cell that producesthe cytokine(s). Certain cytokines are important in drivingpathophysiological changes in arthritic joints, while other cytokinesoffer protective effects against these changes. Cytokines exhibiting aprotective effect include various forms of interleukin (IL) includingIL-4, IL-10 and IL-13; all of these cytokines act in ananti-inflammatory capacity, as an immuno-suppressive agent, or exert animmunostimulatory effect, depending on the target cell. It is alsobelieved that they protect against cartilage breakdown.

Viral IL-10 (vIL-10), another cytokine, is a variant of IL-10 producedby the Epstein Barr virus. This virally encoded gene product is alsoimmuno-suppressive and anti-inflammatory.

Growth factors are types of cytokines that are anti-arthritic in thatthey maintain synthesis of the cartilaginous matrix. Growth factorsinclude, but are not limited to, transforming growth factor (TGF),TGF-β1, TGF-β2 and TGF-β3, fibroblast growth factor (FGF), aFGF andbFGF, insulin-like growth factor (IGF), IGF-1 and IGF-2. While theeffect of certain growth factors is not known, IGF's are known tomaintain the synthesis of the cartilaginous matrix, and promotecartilage repair.

Growth hormone, and at least some of the bone morphogenetic proteins(BMP) are also cytokines. Growth hormone is believed to act by inducinglocal synthesis of IGF-1, although the inventors do not wish to be boundby this mechanism. There are at least nine BMP's; the BMP's are membersof the TGF-β super family. BMP's induce the formation of both bone andcartilage. BMP-2 and BMP-7 (also known as osteogenic protein-1 (OP-1))have shown to be particularly promising in the therapeutic treatment ofconnective tissue disorders, and are therefore the preferred BMP's foruse in the methods of the present invention.

As used herein, the term “cytokine” refers to all of the therapeuticcytokines described above.

CTLA4 is a surface molecule found on T-cells, which binds to acounter-ligand known as B7 on the surface of antigen-presenting cells(APC's). In its soluble form, CTLA4 binds to B7 and thereby prevents B7from interacting with a co-stimulatory molecule known as CD28 on thesurface of the T-cell. When B7-CD28 interactions are blocked in thisway, T-cell activation and hence the immune response is prevented. Thereis evidence that this process can induce immune tolerance. CTLA4 istypically used in soluble form.

Fas ligand (FasL) is a cell surface protein that binds to anotherprotein, called Fas, found on the surface of other cells, includinglymphocytes. When FasL binds to Fas, the cell expressing Fas undergoesapoptosis. Soluble FasL may also induce apoptosis and may be used tokill lymphocytes, as well as other Fas⁺ cells in synovium.

Various anti-adhesion molecules are also within the scope of the presentinvention. These molecules function by inhibiting cell-cell andcell-matrix interactions and have anti-inflammatory properties. Examplesof such proteins, including their fragments and derivatives, are solubleICAM-1 and soluble CD44.

The use of free radical antagonists is also within the scope of thepresent invention. These antagonists function to prevent the deleteriouseffects of free radical formation within the afflicted joint. Examplesinclude but are not limited to the superoxide dismutase and proteins orprotein fragments which inhibit NO and NO synthase.

Preferred genes for use in the present invention for eliciting atherapeutic and/or prophylactic benefit in a host include IRAP, sIL-1RI,sIL-1RII, sTNF-αRI, sTNF-αRII, TIMP-1, TIMP-2, TIMP-3, TIMP-4, PAls,serpins. IL-4, IL-10, IL-13, IGF-1, IGF-2, vIL-10, CTLA4, BMP-1, BMP-2,BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, FasL and their derivative forms. Useof other therapeutic genes is also within the scope of the presentinvention.

The scope of the present invention includes the use of one or more ofthe above-recited therapeutic genes in the therapeutic or prophylactictreatment of a connective tissue disorder. Genes encoding for more thanone protein can be introduced through the same vector, as describedbelow, or can be introduced through the use of different vectors, witheach vector containing a different gene of interest. An unexpecteddiscovery of the present invention is that the use of two or more genestogether produces an enhanced therapeutic benefit. Particularlypreferred for use together are genes encoding for sTNF-αR and sIL-1R.Other gene combinations are within the scope of the present invention aswell. When administering two or more different genes through two or moredifferent vectors or other means of delivery, each of the delivery meanscan be introduced simultaneously or can be introduced in succession. Ifin succession, introduction of the second, third, or greater genes ispreferably done immediately following introduction of the first gene, toensure that the highest levels of expression of each gene are achievedin the host at the same time.

Numerous methods for introducing one or more of the above-described DNAsequences to the host can be used. For example, both viral and nonviralvectors can be prepared, which contain the DNA sequence(s) of interest.A preferred embodiment of this invention includes employing as the viralvector a retroviral vector. More specifically, this method includesemploying as the retroviral vector at least one material selected fromthe group consisting of MFG and pLJ. An MFG vector is a simplifiedMoloney murine leukemia virus vector (MoMLV) in which the DNA sequencesencoding the pol and env proteins have been deleted so as to render itreplication defective. An MFG vector can be prepared that contains oneDNA sequence of interest. Two (DFG), three (TFG) or even more DNAsequences of interest can be included in the MoMLV. Thus, DFG and TFGare forms of MFG having multiple genes. For ease of reference, the termMFG, as used herein, includes any singular or multi-gene form of thevector. A pLJ retroviral vector is also a form of the MoMLV and is morefully described by Korman et al., Proc. Nat'l Acad. Sci., 84:2150-2154(1987), which description is hereby incorporated by reference.

In a preferred embodiment of this invention, a DNA sequence encoding ahuman interleukin-1 receptor antagonist protein (IRAP) or biologicallyactive derivatives or fragments thereof is introduced to the host. TheDNA sequence encoding IRAP or a biologically active derivatives orfragments thereof may be delivered to the connective tissue of amammalian host by any combination of various vector strategies andtransduction techniques disclosed throughout this specification. Apreferred method of delivering IRAP to a target joint space involvesdelivery of the IRAP gene to the synovial lining of a mammalian hostthrough use of the MFG retroviral vector with the ex vivo techniquedisclosed within this specification. A DNA sequence of interest encodinga functional IRAP protein or protein fragment is subcloned into aretroviral vector of choice, the recombinant vector is then grown toadequate titers and used to infect in vitro cultured synovial cells, andthe transduced synovial cells, preferably autografted cells, aretransplanted into the joint of interest, preferably by intra-articularinjection.

Other preferred embodiments of this invention include employing aretroviral vector selected from the group consisting of MFG and pLJ anda DNA sequence encoding soluble interleukin-1 receptor, a soluble TNF-αreceptor and vIL-10.

Any of the DNA sequences and biologically active derivatives orfragments described above can also be introduced to the host by methodsother than retroviral vectors. In another embodiment of this invention,a method is provided for introducing at least one gene encoding aproduct into at least one target cell of a mammalian host for use intreating the mammalian host which comprises employing recombinanttechniques to produce a viral vector containing the gene encoding forthe product and infecting the target cell of the mammalian host usingthe viral vector containing the gene coding for the product, wherein theviral vector is at least one vector selected from the group consistingof an adeno-associated virus, an adenovirus, and a herpes virus, such asherpes simplex type-1 or herpes simplex type-2.

Yet another method of introducing at least one gene encoding a productinto at least one target cell of a mammalian host for use in treatingthe mammalian host includes employing non-viral means for introducingthe gene encoding for the product into the target cell. This methodincludes employing non-viral means selected from the group consisting ofat least one liposome, Ca₃(PO₄)₂, electroporation, and DEAE-dextran.Direct injection of naked DNA can also be used. The liposome can be amaterial selected from the group consisting of DC-chol, SF-chol andnumerous others known to those skilled in the art. It will be understoodthat these non-viral means for introducing the gene encoding for theproduct into the connective tissue cell are non-infectious deliverysystems. An advantage of the use of a non-infectious delivery system isthe elimination of insertional mutagenesis and virally induced disease.It will be appreciated by those skilled in the art that the viralvectors employing a liposome are not limited by cell division as isrequired for the retroviruses to effect infection and integration oftarget cells.

Biological means can also be used to deliver DNA sequences to targetcells. A virus, preferably a pseudo-type retrovirus, can be altered sothat it is capable only of delivery and maintenance within a targetcell, but not retaining an ability to replicate within the target cellor tissue. One or more DNA sequences are introduced to the altered viralgenome, so as to produce a viral genome that acts like a vector, and canbe inserted into a host so that the product of interest will besubsequently expressed.

High levels of collagenase and other tissue metalloproteinases, such asstromelysin and gelatinase can be expressed in the presence of IL-1within connective tissue. Collagenase, stromelysin, and gelatinase areinhibited by the proteins TIMP-1, -2, -3 and -4 (“Tissue Inhibitor ofMetalloProteinases”). Therefore, another preferred embodiment of thisinvention includes providing the method employing viral or non-viralmeans which includes employing one or more forms of TIMP as theproteinase inhibitor. A gene encoding a TIMP protein or biologicallyactive derivative or fragment threat could be delivered to the targetconnective tissue by any combination of means disclosed in thisspecification.

After effecting the infection of the target cell but before thetransplanting of the infected cell into the mammalian host, thetransduced target cells can be stored. It will be appreciated by thoseskilled in the art that the transduced target cells may be stored frozenin 10 percent DMSO in liquid nitrogen.

Another embodiment of this invention includes a method of introducing atleast one gene encoding a product into at least one target cell of amammalian host for use in treating the mammalian host as described abovebut by effecting the infection of the cell in vivo by introducing theDNA sequence coding for the product directly into the mammalian host.Preferably, this method includes effecting the direct introduction ofthe DNA sequence into the mammalian host by intraarticular injection.This method includes employing the method to substantially prevent adevelopment of arthritis in a mammalian host having a highsusceptibility of developing arthritis. This method also includesemploying the method on an arthritic mammalian host for therapeutic use,such as to repair and regenerate connective tissue.

Another preferred method of the present invention involves direct invivo delivery of the IRAP gene to the synovial lining of a mammalianhost through use of either an adenovirus vector, adeno-associated virus(AAV) vector or herpes-simples virus (HSV) vector. A DNA sequence ofinterest encoding a functional IRAP protein or protein fragment issubcloned into the viral vector, the IRAP containing viral vector isthen grown to adequate titers, and directed into the joint space,preferably by intra-articular injection. A retroviral-IRAP construct,such as MFG-IRAP may also be utilized to directly target previouslyinflamed connective tissue cells within the joint space.

Direct intraarticular injection of a DNA sequence containing the gene ofinterest into the joint results in transfection of the recipient targetcells and hence bypasses the requirement of removal, in vitro culturing,transfection, selection, as well as transplanting the DNA vectorcontaining—target cells to promote stable expression of the heterologousgene of interest. Methods of presenting the DNA molecule to the targetcells include, but are not limited to, encapsulation of the DNA moleculeinto cationic liposomes, subcloning the DNA sequence of interest in aviral vector, such as a retroviral vector, as described throughout thisspecification, or the direct injection of the DNA molecule itself intothe joint. The DNA molecule, regardless of the form of presentation tothe knee joint, is preferably presented as a vector molecule, either asrecombinant viral DNA vector molecule, a retroviral vector or arecombinant DNA plasmid vector molecule. Expression of the heterologousgene of interest is ensured by inserting a promoter fragment active ineukaryotic cells directly upstream of the coding region of theheterologous gene. One of the ordinary skill in the art may utilizeknown strategies and techniques of vector construction to ensureappropriate levels of expression subsequent to entry of the DNA moleculeinto the synovial tissue. In vivo delivery of various viral andnon-viral vectors to the rabbit knee joint are described in Example XV.

Both the ex vivo and in vivo methods of the invention can be used in thetherapeutic treatment of patients suffering from one or more of thesymptoms associated with joint pathologies, and in the repair and/orregeneration of connective tissue effected by such pathologies. Any ofthe methods of delivery of one or more genes of interest can be used inconjunction with either the in vivo or ex vivo methodologies. All of themethods can also be used prophylactically, to prevent or retard onset ofthe symptoms of connective tissue disorder in patients susceptible tosuch disorders.

The methods of the present invention provide a means for introduction ofone or more products of interest to the host. These products of interestare generally known in the art as being effective against the symptomsof connective tissue disorders. The amount of each product, in the formof the DNA sequence encoding the product, to introduce will vary frompatient to patient depending on such factors as the size of the patient,the joint affected, the severity of the connective tissue disorder, thegene being used and whether the method is being used therapeutically orprophylactically. Therapeutic responses are typically seen based upondelivery of a vector or other delivery vehicle sufficient to give geneexpression in the high pico- to low nanogram range. One skilled in theart can determine the amount of vector or other delivery means toadminister to a patient to achieve these levels of expression based uponthe factors listed above. Introduction of vectors, such as a retroviralvector, in normal titer (about 10⁵ cfu/ml) is typically sufficient buthigh titer concentrations (equal to or greater than about 10⁷ cfu/ml)are preferred.

A further embodiment of this invention provides for an animal model tostudy connective tissue pathologies and indices of systemicinflammation. This model utilizes either ex vivo or in vivo delivery ofat least one gene or DNA sequence of interest encoding a product into aleast one cell of a connective tissue of a mammalian host. Examples ofjoint pathologies which can be studied in the present invention include,but are not limited to, joint pathologies such as leukocytosis,synovitis, cartilage breakdown, suppression of cartilage matrixsynthesis, edema, inflammation of the eyes, arteritis and rheumatoidnodules. Examples of indices of systemic inflammation include, but arenot limited to, elevated erythrocyte sedimentation rate, fever, weightloss and increases in blood levels of C-reactive protein and IL-6.

A particular embodiment of the present invention which relates to suchan animal model is utilization of the ex vivo based delivery of a DNAsequence encoding human IL-1β gene to the synovial lining of the rabbitknee. In this embodiment, the human IL-1β gene is subcloned into the MFGretroviral vector by known methods, resulting in MFG-IL-1β. Thisrecombinant retroviral construct is used to transduce autologoussynovial cells cultured in vitro. These transduced cells are thendelivered to the rabbit knees as described throughout thisspecification. Delivery of the human IL-1β gene to the synovial liningof the rabbit knee in this fashion causes a severe, chronic,monarticular arthritis. Pathologies include leukocytosis, synovitis,cartilage breakdown and suppression of cartilage matrix synthesis.Various systemic indices of inflammation are also effected, including anincreased erythrocyte sedimentation rate, fever and weight loss. Thisprocedure can also be carried out using any other retroviral vector,viral vector, or non-viral or biological means.

In another example of this particular embodiment of the presentinvention, the human IL-1β gene is subcloned into a DNA plasmid vector,downstream of a CMV promoter. This CMV-IL-1β plasmid construct isassociated with liposomes and delivered to a target joint space, such asdescribed in Example X. Forty eight hours subsequent to injection 100 pgof hIL-1β was recovered from the knee joint area, demonstrating theefficacy of the present methods in delivering hIL-1β to a joint ofinterest.

An animal model as described and exemplified in this specificationmeasures the ability of various gene therapy applications disclosedthroughout this specification to withstand challenges from knowncausative agents (such as IL-1β) of joint pathologies and inflammatoryside effects.

A method to produce an animal model for the study of connective tissuepathology is also contemplated by the present invention. As will beunderstood by those skilled in the art, over-expression of interleukin-1in the joint of a mammalian host is generally responsible for theinduction of an arthritic condition. This invention provides a methodfor producing an animal model using the above described gene transfertechnology of this invention. Preferably, the method of this inventionprovides a method for producing such an animal model for arthritis. Forexample, constitutive expression of interleukin-1 in the joint of arabbit following the method of gene transfer provided for by thisinvention leads to the onset of an arthritic condition. It will beappreciated by those skilled in the art that this rabbit model issuitable for use for the testing of therapeutic agents. This methodincludes introducing at least one gene encoding a product into at leastone target cell of a mammalian host comprising (a) employing recombinanttechniques to produce a viral vector which contains the gene encodingfor the product and (b) infecting the target cell of the mammalian hostusing the viral vector containing the gene coding for the product foreffecting the animal model.

Any gene known to contribute to one or more of the symptoms ofconnective tissue disorders can be used in the animal model. As with thetherapeutic treatment methodology, more than one gene can be introduced.Genes suitable for use in the animal model methods of the presentinvention, therefore, include any genes which cause such a symptom,including but not limited to various forms of interleukin such as IL-1α,IL-1β, IL-2, IL-7 IL-8, IL-12, IL-15 and IL-17, TNF-α, TNF-β, iNOS andproteinases including but not limited to aggrecanase, or a matrixmetalloproteinase selected from the group consisting of at least onecollagenase, gelatinase and stromelysin. Inducible nitric oxide synthase(iNOS or NOSII) is an enzyme found in athritic joints, which catalyzesthe formation of the radical nitric oxide (NO).

Any biologically active derivatives or fragments of these genes can alsobe used. One skilled in the art can test the biological activity of suchderivatives or fragments by evaluating their ability to contribute toone or more of the deleterious symptoms associated with connectivetissue disorders.

Non-viral means for introducing at least one gene encoding a productinto at least one cell of a connective tissue of a mammalian host canalso be used in the animal model embodiment. The non-viral means isselected from the group consisting of at least one liposome, Ca₃(PO₄)₂,electroporation, DEAE-dextran and injection of naked DNA. The genesdescribed above can be introduced by any of these non-viral means.

Any of the viral or non-viral means described in conjunction with thetherapeutic method can be used to effect delivery of the DNA sequence orsequences of interest in the animal model. Also, any of the connectiveor non-connective tissue cells can be targeted in the animal model, asdescribed above for the therapeutic methods. It will be appreciated bythose skilled in the art that introduction of any of the deleteriousgenes listed above will result in conditions mimicking those seen in ananimal suffering from a connective tissue disorder. The afflicted animalcan then be used to study potential methods for therapeutically treatingsuch connective tissue disorders experienced by humans. Thus, the animalmodel of the present invention provides a correlatable means of studyingconnective tissue disorders.

EXAMPLES

The following examples are intended to illustrate the present invention,and should not be construed as limiting the invention in any way.

Example I

Packaging of AAV

The only cis-acting sequences required for replication and packaging ofrecombinant adeno-associated virus (AAV) vector are the AAV terminalrepeats. Up to 4 kb of DNA can be inserted between the terminal repeatswithout effecting viral replication or packaging. The virus rep proteinsand viral capsid proteins are required in trans for virus replication asis an adeno-associated virus helper. To package a recombinant AAVvector, the plasmid containing the terminal repeats and the therapeuticgene is co-transfected into cells with a plasmid that expresses the repand capsid proteins. The transfected cells are then infected withadeno-associated virus and virus isolated from the cells about 48-72hours post-transfection. The supernatants are heated to about 56°Centigrade to inactivate the adeno-associated virus, leaving an activevirus stock of recombinant AAV.

Example II

Electroporation

The connective tissue cells to be electroporated are placed into Herpesbuffer saline (HBS) at a concentration of about 10⁷ cells per ml. TheDNA to be electroporated is added at a concentration of about 5-20 ug/mlof HBS. The mixture is placed into a cuvette and inserted into thecuvette holder that accompanies the Bio-RAD electroporation device (1414Harbour Way South, Richmond, Calif. 94804). A range between about 250and 300 volts at a capacitance of about 960 ufarads is required forintroduction of DNA into most eukaryotic cell types. Once the DNA andthe cells are inserted into the Bio-RAD holder, a button is pushed andthe set voltage is delivered to the cell-DNA solution. The cells areremoved from the cuvette and replated on plastic dishes.

Example III

The cDNA encoding the human interleukin-1 receptor antagonist (IRAP) wasinserted into the NcoI and BamHI cloning sites of the retroviral vectorMFG as shown in FIG. 1. Specifically, a PstI to BamHI fragment from theIRAP cDNA was linked to a synthetic oligonucleotide adapter from theNcoI site (representing the start site of translation for IRAP) to thePstI site (approximately 12 base pairs downstream from the NcoI site) tothe MFG backbone digested at NcoI and BamHI in a three part ligationreaction. This three part ligation involving a synthetic oligo and twoDNA fragments is well known by those skilled in the art of cloning. LTRmeans long terminal repeats, 5′SD means 5′ splice donor, 3′SA means 3′splice acceptor. The straight arrow and the crooked arrow in FIG. 1represent unspliced and spliced messenger RNAs respectively. IRAP isencoded by the spliced message.

FIG. 2 shows the cDNA encoding the human interleukin-1 receptorantagonist protein (IRAP) with a selectable neo gene marker. FIG. 3shows a low power micrograph of synovium recovered from the knee of arabbit one month after intra-articular injection of Lac Z⁺, neo⁺synoviocytes. Tissue was stained histochemically for the presence ofbeta-galactosidase. This micrograph counterstained with eosin revealedan area of intensely stained, transplanted cells demonstrating thatthese cells have colonized the synovial lining of the recipient joint.

Example IV

Animal Models

The methods of this invention of transferring genes to the synovia ofmammalian joints permit the production and analysis of joint pathologiesthat were not previously possible. This is because the only other way ofdelivering potentially arthritogenic compounds to the joint is byintra-articular injection. Not only are such compounds quickly clearedfrom joints, but the effects of bolus injections of these compounds donot accurately mimic physiological conditions where they are constantlyproduced over a long period of time. In contrast, the gene transfertechnologies of this invention permit selected proteins of known orsuspected involvement in the arthritic process to be expressedintraarticularly over an extended period of time, such as for example,at least a three month period. The animal models of this inventiontherefore permit the importance of each gene product to the arthriticprocess to be evaluated individually. Candidate genes include, but arenot restricted to, those coding for cytokines such as interleukin-1alpha (IL-α), IL-1 beta (IL-1β) and TNF-alpha, (TNF-α) and matrixmetalloproteinases such as collagenases, gelatinases and stromelysins.

Additionally, the gene transfer techniques of this invention aresuitable for use in the screening of potentially therapeutic proteins.In this use, the animal models of the invention are initiated in jointswhose synovia express one or more genes coding for potentialanti-arthritic proteins. Candidate proteins include, but are notrestricted to, inhibitors of proteinases such as the tissue inhibitor ofmetalloproteinases, and cytokines such as transforming growthfactor-beta (TGF-β).

Example V

Method for Using Synoviocytes as a Delivery System for Gene Therapy

Rabbits are killed by intravenous injection of 4 ml nembutol, and theirknees quickly shaved. Synovia are surgically removed from each kneeunder aseptic conditions, and the cells removed from their surroundingmatrix by sequential digestion with trypsin and collagenase (0.2% w/v inGey's Balanced Salt Solution) for about 30 minutes and about 2 hours,respectively. The cells recovered in this way are seeded into 25 cm²culture flasks with about 4 ml of Ham's F₁₂ nutrient medium supplementedwith 10% fetal bovine serum, about 100 U/ml penicillin and about 100μg/ml streptomycin, and incubated at about 37° in an atmosphere of 95%air, 5% CO₂. Following about 3-4 days incubation, the cells attainconfluence. At this stage, the culture medium is removed and the cellsheet washed twice with approximately 5 mls of Gey's Balanced SaltSolution to remove non-adherent cells such as lymphocytes. The adherentcells are then treated with trypsin (0.25% w/v in balanced saltsolution). This treatment detaches the fibroblastic, Type Bsynoviocytes, but leaves macrophages, polymorphionuclear leukocytes andthe Type A synoviocytes attached to the Culture vessel. The detachedcells are recovered, re-seeded into 25 cm² culture vessels at a 1:2split ratio, medium is added and the culture returned to the incubator.At confluence this procedure is repeated.

After the third such passage, the cells are uniformly fibroblastic andcomprise a homogeneous population of Type B synoviocytes. At this stage,cells are infected with the retroviral vector.

Following infection, cells are transferred to fresh nutrient mediumsupplemented with about 1 mg/ml G418 (GIBCO/BRL, P.O. Box 68, GrandIsland, N.Y. 14072-0068) and returned to the incubator. Medium ischanged every three days as neo cells die and the neo⁺ cells proliferateand attain confluency. When confluent, the cells are trypsinized andsubcultured as described above. One flask is set aside for staining withX-gal to confirm that the neo⁺ cells are also Lac Z⁺. When thesubcultures are confluent, the medium is recovered and tested for thepresence of IRAP, soluble IL-1R or other appropriate gene products ashereinbefore described. Producing synoviocyte cultures are then readyfor transplantation.

The cells are recovered by centrifuging, washed several times byresuspension in Gey's Balanced Salt Solution and finally resuspended ata concentration of about 10⁶-10⁷ cells/ml in Gey's Solution.Approximately 1 ml of this suspension is then introduced into the kneejoint of a recipient rabbit by intra-articular injection. For thispurpose a 1 ml syringe with a 25-gauge hypodermic needle is used.Injection is carried out through the patellar tendon. Experiments inwhich radiopaque dye was injected have confirmed that this methodsuccessfully introduces material into all parts of the joint.

Variations on the disclosed harvesting, culture and transplantationconditions in regard to the numerous examples presented within thisspecification will be evident upon inspection of this specification.Several tangential points may be useful to one practicing the ex vivobased gene therapy portion of the disclosed invention:

-   -   (1) If the yield of synoviocytes from the harvested synovial        tissue is poor, the surgical technique may be at fault. The        synovium has a strong tendency to retract when cut. Therefore,        the inner capsule is grasped firmly, and with it the synovium,        while excising this tissue. A small (about 2 mm) transverse        incision can be made inferiorly, followed by sliding one point        of the forceps into the joint space so that the synovium and        inner capsule are sandwiched between the points of the forceps.        The tissue is then excised without releasing the tissue thus        preventing retraction of the synovium.    -   (2) A two compartment digestion chamber may be used to initially        separate the cells from extracellular debris. In lieu of this        choice, synovial tissue may be digested in a single chamber        vessel and filtered through a nylon monofilament mesh of 45 μm        pore size.    -   (3) When resuspending cells, the smallest amount of medium        possible can be used to prevent formation of clumps of cells,        which are difficult to separate once formed. EDTA in millimolar        amounts can also be used to prevent clumps.    -   (4) During trypsinization, synoviocytes can lose the fusiform        morphology that they possess in adherence, and assume a rounded        shape. The cells initially will detach in clumps of rounded        cells; one may allow the majority of cells to separate from each        other before stopping trypsinization.    -   (5) Synoviocytes may be transduced with multiple transgenes by        use of retroviral vectors containing multiple transgenes or by        sequential transduction by multiple retroviral vectors. In        sequential transduction, the second transduction should be made        following selection, when applicable, and passage after the        first transduction.

(6) As the synovium is a well-innervated structure, intra-articularinjection can be painful, especially if done rapidly. Intra-articularinjection of a 1 ml volume should take 10 to 15 seconds.

(7) In the animal model, the depth of the needle stick should not exceed1 cm during intraarticular injection, and depression of the syringeplunger should meet with little to no resistance. Resistance toadvancement of the syringe plunger indicates that the tip of the needleis not in the joint space.

(8) In the animal model, to retrieve a useful volume of the injectedGey's solution during joint lavage, the needle should not be insertedtoo deeply, otherwise it may penetrate the posterior capsule and maylacerate the popliteal artery. Firm massage of the suprapatellar,infrapatellar, and lateral aspects of the knee during aspiration helpsto increase the amount of fluid recovered; in general, it should bepossible to recover≧0.5 ml of fluid. When knees are badly inflamed,lavage is often difficult because of the presence of large numbers ofleukocytes, fibrin, and other debris in the joint. The animal can beanesthetized or sacrificed and the Gey's solution recorded surgically.

Example VI

The method of Example V for producing generally uniformly fibroblasticcells of a homogeneous population of Type B synoviocytes was followed toeffect growing cultures of lapine synovial fibroblasts. These growingcultures of lapine synovial fibroblasts were subsequently infected withan amphotropic retroviral vector carrying marker genes coding forbeta-galactosidase (Lac Z) and resistance to the neomycin analogue G418(neo⁺). Following infection and growth in selective medium containingabout 1 mg/ml G418, all cells stained positively in a histochemicalstain for beta-galactosidase.

Neo selected cells carrying the Lac Z marker gene were transplanted backinto the knees of recipient rabbits to examine the persistence andexpression of these genes in vivo. Two weeks following transplantation,islands of Lac Z⁺ cells within the synovium of recipient knees wereobserved. This confirmed the ability of the method of this invention tointroduce marker genes into rabbit synovia and to express them in situ.

Example VII

Neo-selected, Lac Z⁺ synoviocytes were recovered from cell culture,suspended in Gey's Balanced Salt Solution and injected intra-articularlyinto the knee joints of recipient rabbits (about 10⁵-10⁷ cells perknee). Contralateral control knees received only a carrier solution. Atintervals up to 3 months following transplant, the rabbits were killedand their synovia and surrounding capsule recovered. Each sample may beanalyzed in three ways. A third of the synovium was stainedhistochemically en masse for the presence of beta-galactosidase. Asecond portion may be used for immunocytochemistry using antibodiesspecific for bacterial beta-galactosidase. The final portion may bedigested with trypsin and collagenase, and the cells thus recoveredcultured in the presence of G418.

Staining of the bulk synovial tissue revealed extensive areas of Lac Z⁺cells, visible to the naked eye. Control synovia remained colorless.Histochemical examination of synovia revealed the presence of islands ofcells staining intensely positive for beta-galactosidase. These cellswere present on the superficial layer of the synovial lining, and wereabsent from control synovia. From such tissue it was possible to growLac Z⁺, neo⁺ cells. Cells recovered from control tissue were Lac Z⁻ anddied when G418 was added to the culture. This indicates that thetransplanted, transduced synovial fibroblasts have successfullyrecolonized the synovia of recipient joints, and continue to express thetwo marker genes, Lac Z and neo. Maintaining intra-articular Lac Z andneo expression in transplanted synoviocytes has been effected for about6 weeks using primary cells and about 2 weeks using the HIG-82 cellline.

Example VIII

Based upon the methods of the hereinbefore presented examples, andemploying standard recombinant techniques well known by those skilled inthe art, the human IRAP gene was incorporated into an MFG vector asshown in FIG. 1. Following the infection of synoviocyte cultures ofrabbit origin with this viral vector, IRAP was secreted into the culturemedium.

Western blotting, well known by those skilled in the art, was carriedout using an IRAP-specific rabbit polyclonal antibody that does notrecognize human or rabbit IL-1 alpha or IL-1 beta, or rabbit IRAP. FIG.4 shows a Western blot which sets forth the production of IRAP by fourcultures of HIG-82 cells infected with MFG-IRAP. Three forms of the IRAPare present: a non-glycosylated form which runs with recombinantstandards, and two larger glycosylated forms. The results of the Westernblotting shown in FIG. 4 demonstrated that IRAP was produced by HIG-82synoviocyte cell line (Georgescu, 1988, In Vitro 24: 1015-1022)following infection with the MFG-IRAP vector of this invention. TheWestern blotting of FIG. 4 shows the IRAP concentration of theconditioned medium is as high as 50 ng/ml. This is approximately equalto 500 ng IRAP/10⁶ cells/day. Lane 1 and Lane 2 of FIG. 4 show that therecipient synovia tissue secrete substantial amounts of HIG-IRAP at 3days (Lane 2) and 6 days (Lane 1). Lane 3 shows human recombinant IRAP.Lane 6 indicates that rabbit synovial cells produce a largerglycosylated version of this molecule after infection with MFG-IRAP.Lane 7 indicates that native rabbit synovial cells do not produce thisglycosylated form.

FIG. 5 shows that medium conditioned by IRAP⁺ synoviocytes blocks theinduction of matrix metalloproteinases in articular chondrocytes exposedto recombinant human IL-1 beta. Chondrocytes normally secrete 1 U/10⁶cells, or less, gelatinase into their culture media. FIG. 5 shows thatwhen to about 5 U/ml or 10 U/ml IL-1 are added, gelatinase productionincreases to over 4 U and 6 U/10⁸ cells, respectively. Addition ofmedium conditioned by MFG-IRAP-infected HIG-82 cells employed by themethod of this invention suppressed gelatinase production by IL-1treated chondrocytes. With 5 U/ml IL-1 (FIG. 5, right panel) inhibitionwas 100% for one culture and 41% for the other. With 10 U/ml IL-1,inhibition was reduced to 38% and 18% (FIG. 5, left panel) as isexpected of a competitive inhibitor. These data demonstrate that theIRAP produced by HIG-82 cells infected with MFG-IRAP is biologicallyactive.

Example IX

This example demonstrates the uptake and expression of Lac Z gene bysynoviocytes using infection by a liposome (lipofection). A six wellplate containing synoviocyte cultures were transduced with the Lac Zgene by lipofection. The content of each well is as follows:

Well 1 Control cells, treated with liposomes alone Well 2 Control cells,treated with DNA alone Well 3 DNA + 150 nmole liposomes Well 4 DNA + 240nmole liposomes Well 5 DNA + 300 nmole liposomes Well 6 DNA + 600 nmoleliposomes

Wells 3-6 containing sub-confluent cultures of synovial fibroblasts weretransfected with 6 ug of DNA complexed with 150-600 nmoles/well of“DC-chol” liposome or in the alternative, with “SF-chol”. Three dayslater, cells were stained histochemically for expression ofbeta-galactosidase (FIG. 6).

Table 1 shows the results of using the liposomes “DC-chol” and “SF-chol”in converting synoviocyte cultures to the Lac Z⁺ phenotype withoutselection. Table 1 sets forth that the “DC-chol” liposome in aconcentration of about 300 nmole/well converted generally 30% of thesynovial cells in synoviocyte cultures to the Lac Z⁺ phenotype withoutselection. Reduced expression was shown in Well 6 for “DC-chol” due tothe toxic effect of the high liposome concentration.

TABLE 1 % Lac Z⁺ Cells Liposome, nmole/well DC-chol SF-chol 150 10 0.5240 22 1.0 300 30 2.8 600 NA 3.5

In another embodiment of this invention, a gene and method of using thisgene provides for the neutralization of interleukin-1. Interleukin-1 isa key mediator of cartilage destruction in arthritis. Interleukin-1 alsocauses inflammation and is a very powerful inducer of bone resorption.Many of these effects result from the ability of interleukin-1 toincrease enormously the cellular synthesis of prostaglandins, and avarious proteinases including collagenase, gelatinase, and stromelysin,a plasmiinogen activator and aggrecanase. The catabolic effects ofinterleukin-1 upon cartilage are exacerbated by its ability to suppressthe synthesis of the cartilaginous matrix by chondrocytes. Interleukin-1is present at high concentrations in synovial fluids aspirated fromarthritic joints and it has been demonstrated that intra-articularinjection of recombinant interleukin-1 in animals causes cartilagebreakdown and inflammation.

Interleukin-1 exists as several species, such as unglycosylatedpolypeptide of 17,000 Daltons. Two species have previously been cloned,interleukin-1 alpha and interleukin-1 beta. The alpha form has a pI ofapproximately 5, and the beta form has a pI around 7. Despite theexistence of these isoforms, interleukin-1 alpha and interleukin-1 betahave substantially identical biological properties and share common cellsurface receptors. The type I interleukin-1 receptor is a 80 kDa(kilodalton) glycoprotein and contains an extracellular, interleukin-1binding portion of 319 amino acids which are arranged in threeimmunoglobulin-like domains held together by disulfide bridges as shownin FIG. 7. A 21 amino acid trans-membrane domain joins the extracellularportion to the 217 amino acid cytoplasmic domain. FIGS. 8A-8C show theamino acid and nucleotide sequence of the human and mouse interleukin-1receptors. In FIG. 8B, the 21 amino acid trans-membrane region of theinterleukin-1 receptor is marked by the thicker solid line. In FIGS. 8Aand 8B, the position of the 5′ and 3′ oligonucleotides for PCR aremarked by thinner short lines, respectively. The lysine amino acid just5′ to the trans-membrane domain to be mutated to a stop codon is markedby a solid circle in FIG. 8B.

Synovium is by far the major, and perhaps the only, intraarticularsource of interleukin-1 in the arthritic joint. Synovia recovered fromarthritic joints secrete high levels of interleukin-1. Both the residentsynoviocytes and infiltrating blood mononuclear cells within thesynovial lining produce interleukin-1.

The present invention provides a method of using in vivo a gene codingfor a truncated form of the interleukin-1 receptor which retains itsability to bind interleukin-1 with high affinity but which is releasedextracellularly and therefore inactive in signal transduction. Thebinding of this truncated and modified receptor to interleukin-1inhibits the intraarticular activity of interleukin-1.

This method of using a gene encoding the extracellular interleukin-1binding domain of an interleukin-1 receptor that is capable of bindingto and neutralizing interleukin-1 includes employing a retroviral vectorcarrying a truncated interleukin-1 receptor gene which encodes atruncated and soluble active form of the receptor. The expression of thenovel interleukin-1 receptor gene is controlled by regulatory sequencescontained within the vector that are active in eukaryotic cells. Thisrecombinant viral vector is transfected into cell lines stablyexpressing the viral proteins in trans required for production ofinfectious virus particles carrying the recombinant vector. These viralparticles are used to deliver the recombinant interleukin-1 receptor tothe recipient synovial cells by direct virus infection in vivo.

The soluble human interleukin-1 receptor to be inserted into theretroviral vector may be generated by a polymerase chain reaction (PCR).An oligonucleotide complementary to the 5′ leader sequence of the humaninterleukin-1 receptor (GCGGATCCCCTCCTGAGAAGCT; SEQ ID NO: 5) and anoligonucleotide complementary to a region just upstream from thetransmembrane domain of the interleukin-1 receptor(GCGGATCCCATGTGCTACTGG; SEQ ID NO: 6) are used as primers for PCR. Theprimer for the region of the interleukin-1 receptor adjacent to thetrans-membrane domain contains a single base change so that the lyscodon at amino acid 336 (AAG) is changed to a stop codon (TAG). Byinserting a translation stop codon just upstream from the transmembranedomain, a truncated form of interleukin-1 receptor that is secreted bythe cell is generated. A BamHI recognition sequence (GGATCC) is added tothe 5′ end of the PCR primers, and following amplification, theresulting interleukin-1 receptor fragment is cloned into a BamHI site. AcDNA library from human T-cells is used as a source for theinterleukin-1 receptor cDNA. To amplify the appropriate region of theinterleukin-1 receptor from the cDNA library, the complementary primersare added to the DNA and 50 cycles of annealing primer extension anddenaturation are performed using a thermocycler and standard PCRreaction conditions well known by those persons skilled in the art.Following amplification of the interleukin-1 soluble receptor using thePCR process, the resulting fragment is digested with BamHI and insertedinto the pLJ retroviral vector. The pLJ retroviral vector is availablefrom A. J. Korman and R. C. Mulligan. See also Proc. Nat'l. Acad. Sci.,84:2150-2154 (April 1987) co-authored by Alan J. Korman, J. DanielFrantz, Jack L. Strominger and Richard C. Mulligan. Restriction analysiswas performed to determine the correct orientation of the insert. Itcould also be cloned into MFG.

The retrovirus vector carrying the truncated interleukin-1 receptor istransferred into the CRIP (Proc. Nat'l. Acad. Sci. Vol. 85, pp.6460-6464 (1988), O. Danos and R. C. Mulligan) packaging cell line usinga standard Ca₃(PO₄)₂ transfection procedure and cells wherein the viralvector is stably integrated and is selected on the basis of resistanceto the antibiotic G418. The viral vector containing the neomycinresistant (neo-r) gene is capable of imparting resistance of the cellline to G418. The CRIP cell line expresses the three viral proteinsrequired for packaging the vector viral RNAs into infectious particles.Moreover, the viral particles produced by the CRIP cell line are able toefficiently infect a wide variety of mammalian cell types includinghuman cells. All retroviral particles produced by this cell line aredefective for replication but retain the ability to stably integrateinto synovial cells thereby becoming an heritable trait of these cells.Virus stocks produced by this method are substantially free ofcontaminating helper-virus particles and are also non-pathogenic.

More specifically, the truncated interleukin-1 gene can be inserted intoa retroviral vector under the regulation of a suitable eukaryoticpromoter such as the retroviral promoter already contained within thegene transfer vector, such as for example, the pLJ vector shown in FIG.9. FIG. 9 shows the structure of the pLJ interleukin receptor retroviralvector and partial restriction endonuclease map. Reference numeral 10shows the interleukin-1 receptor inserted into a retroviral vector.Reference numeral 12 indicates long terminal repeats (LTR's) at each endof the structure of the pLJ interleukin receptor retroviral vector shownin FIG. 8. These LTR's regulate the viral transcription and expressionof the interleukin-1 receptor. Bacterial gene encoding resistance to theantibiotic neomycin (neo-r) is shown at reference numeral 16. The SimianVirus 40 enhancer promoter (SV 40) is indicated at reference numeral 18,and regulates the expression of the neo-r gene. Reference numbers 20 and22, respectively, show the sites wherein the resulting interleukinreceptor fragment is cloned. It will be understood by those personsskilled in the art that other vectors containing different eukaryoticpromoters may also be utilized to obtain a generally maximal level ofinterleukin-1 receptor expression. The vectors containing the truncated,and modified interleukin-1 receptor will be introduced into a retroviralpackaging cell line (CRIP) by transfection and stable transformantsisolated by selection for the expression of the neomycin resistance genealso carried by the pLJ vector. The CRIP cell line expresses all theproteins required for packaging of the exogenous retroviral RNA. Viralparticles produced by the G418-selected CRIP cell lines will carry arecombinant retrovirus able to infect mammalian cells and stably expressthe interleukin-1 truncated receptor. The viral particles can be used toinfect synovial cells directly in vivo by injecting the virus into thejoint space or alternatively in vitro as part of ex vivo transplantmethods.

Another embodiment of this invention provides a method for using thehereinbefore described viral particles to infect in culture synovialcells obtained from the lining of the joint of a mammalian host. Theadvantage of the infection of synovial cells in culture is that infectedcells harboring the interleukin-1 receptor retroviral construct can beselected using G418 for expression of the neomycin resistance gene. Theinfected synovial cells expressing the interleukin-1 receptor can thenbe transplanted back into the joint by intra-articular injection. Thetransplanted cells will express high levels of soluble interleukin-1receptor in the joint space thereby binding to and neutralizingsubstantially all isoforms of interleukin-1, including interleukin-1alpha and interleukin-1 beta.

The method used for transplantation of the synovial cells within thejoint is a routine and relatively minor procedure used in the treatmentof chronic inflammatory joint disease. Although synovium can berecovered from the joint during open surgery, it is now common toperform synovectomies, especially of the knee, through the arthroscope.The arthroscope is a small, hollow rod inserted into the knee via asmall puncture wound. In addition to permitting the intraarticularinsertion of a fibre-option system, the arthroscope allows access tosurgical instruments, such that synovial tissue can be removedarthroscopically. Such procedures can be carried out Linder “spinal”anesthetic and the patient allowed home the same day. In this mannersufficient synovium can be obtained from patients who will receive thisgene therapy.

The synovial cells (synoviocytes) contained within the excised tissuemay be aseptically recovered by enzymic digestion of the connectivetissue matrix. Generally, the synovium is cut into pieces ofapproximately 1 millimeter diameter and digested sequentially withtrypsin (0.2% w/v in Gey's Balanced Salt Solution) for 30 minutes at 37°Centigrade, and collagenase (0.2% w/v in Gey's Balanced Salt Solution)for 2 hours at 37° Centigrade. Cells recovered from this digestion areseeded into plastic culture dishes at a concentration of 10⁴-10⁵ cellsper square centimeter with Ham's F₁₂ medium supplemented with 10% fetalbovine serum and antibiotics. After 3-7 days, the culture medium iswithdrawn. Non-adherent cells such as lymphocytes are removed by washingwith Gey's Balanced Salt Solution and fresh medium added. The adherentcells can now be used as they are, allowed to grow to confluency ortaken through one or more subcultures. Subcultivating expands the cellnumber and removes non-dividing cells such as macrophages.

Following genetic manipulation of the cells thus recovered, they can beremoved from the culture dish by trypsinizing, scraping or other means,and made into a standard suspension. Gey's Balanced Salt Solution orother isotonic salt solutions of suitable composition, or salinesolution are suitable carriers. A suspension of cells can then beinjected into the recipient mammalian joint. Intra-articular injectionsof this type are routine and easily carried Out in the doctor's office.No surgery is necessary. Very large numbers of cells can be introducedin this way and repeat injections carried out as needed.

Another embodiment of this invention is the gene produced by thehereinbefore described method of preparation. This gene carried by theretrovirus may be incorporated in a suitable pharmaceutical carrier,such as for example, buffered physiologic saline, for parenteraladministration. This gene may be administered to a patient in atherapeutically effective dose. More specifically, this gene may beincorporated in a suitable pharmaceutical carrier at a therapeuticallyeffective dose and administered by intra-articular injection. Therefore,the preferred mode regarding the ex vivo method of delivery is theremoval of the patients connective tissue (e.g., synovia), in vitroculture of this connective tissue, transduction of the DNA sequence ofinterest, followed by the above-mentioned manipulation prior to deliveryto the afflicted joint of the patient.

In another embodiment of this invention, this gene may be administeredto patients as a prophylactic measure to prevent the development ofarthritis in those patients determined to be highly susceptible ofdeveloping this disease. More specifically, this gene carried by theretrovirus may be incorporated in a suitable pharmaceutical carrier at aprophylactically effective dose and administered by parenteralinjection, including intraarticular injection.

Example X

Fifty micrograms of a DNA plasmid vector molecule containing theinterleukin-1 beta coding sequence ligated downstream of the CMVpromoter was encapsulated within cationic liposomes, mixed with Gey'sbiological buffer and injected intraarticularly into the knee joints ofa rabbit. Forty eight hours subsequent to injection one nanogram ofinterleukin-1 beta was recovered from the knee joint area. Therefore,injection of the DNA containing liposome solution within the region ofthe synovial tissue prompted fusion of the liposomes to the synovialcells, transfer of the DNA plasmid vector into synovial cells andsubsequent expression of the IL-1 beta gene. Additionally, it ispossible to inject non-encapsulated (i.e., naked) DNA into the jointarea and monitor transfection of the DNA vector into the synovial cellsas determined by subsequent expression of the IL-1 beta gene in synovialcells. Therefore; either method may be utilized as a plausiblealternative to the in vitro manipulation of synovia also exemplified inthe present invention.

Example XI

The in vivo biological activity of the MFG-IRAP construct was tested asthe ability to suppress the effects of IL-1β. Rabbit knees were injectedwith recombinant human IL-1β, known to cause an increased concentrationof leukocytes within the afflicted joint space. Introduction ofMFG-IRAP/HIG-82 cells into rabbit knees strongly suppresses IL-1βproduction of leukocytes to the afflicted joint space. In contrast,control HIG-82 cells do not suppress the leukocyte infiltration to thejoint space challenged with IL-1β (see FIG. 10). Inhibition is greatestat the lowest doses of human recombinant IL-1β (hrIL-1β), as expected bythe competitive mechanism through which IRAP antagonizes IL-1.Therefore, this rabbit model confirms that in vivo, intra-articularexpression of IRAP is biologically active and can protect the joint frominflammation provoked by IL-1.

Example XII

This example further evaluates ex vivo delivery into rabbit knee jointsof the MFG-IRAP construct. As known, IRAP is an acidic glycoprotein ofapproximately 25 kDa that functions as a natural antagonist of thebiological actions of interleukin-1 (IL-1) by binding to IL-1 receptors.Unlike IL-1 IRAP has no agonist activity, instead acting as acompetitive inhibitor of the binding of IL-1.

This example shows that in Vivo expression of IRAP by geneticallymodified synovial cells inhibits IL-1β-induced leukocyte infiltrationinto the joint space, synovial thickening and hypercellularity, and lossof proteoglycans from articular cartilage.

As mentioned within this specification, the preferred lode of treating apatient through the ex vivo route will be by transplanting geneticallymodified autologous synovial cells by intra-articular injection.However, HIG-82 cells, easily maintained in culture, were used for theseexperiments to show that intra-articularly expressed IRAP is effectivein inhibiting the physiological sequelae of intra-articularly injectedIL-1.

MFG-IRAP/HIG-82 cells or control (uninfected HIG-82) cells, weretransplanted into rabbit knees by intra-articular injection by themethods disclosed within this specification. Briefly, cultures of thesecells were infected with MFG-IRAP. Media conditioned for 3 days byinfected MFG-IRAP/HIG 82 cells were assayed for human IRAP by ELISAassay using a commercial kit (R&D Systems, Minneapolis, Minn., USA) andfound to contain approximately 500 ng IRAP/10⁶ cells. Western blottingconfirmed the presence of human IRAP of size 22-25 kDa. HIG-IRAP cellswere trypsinized, suspended in Gey's balanced salt solution and 1 ml ofsuspension, containing 10⁷ cells, was injected intra-articularly intothe left knee joints of New Zealand White rabbits (2.5 kg). Thecontralateral control knees received a similar injection ofuntransducecd HIG-82 cells.

Three days following transplantation of the cells, knee joints werechallenged by various doses of a single intra-articular injection ofhuman recombinant IL-1β dissolved in 0.5 ml Gey's solution. Controlknees were injected with 0.5 ml of Gey's solution.

Eighteen hours after injection of hrIL-1β, rabbits were killed and theknee joints evaluated histopathologically and for expression of IRAP.Each joint was first lavaged with 1 ml Gey's solution containing 10 mMEDTA. Cal counts in these washings were performed with a hemocytometer.An aliquot was removed for cytospinning and staining with ‘DiffQuick’(Baxter Scientific Products) before examination under light microscopy.Washings were then centrifuged. Supernatants were removed for IRAP ELISAand for the determination of glycosaminoglycan (GAG) concentrations asan index of cartilage breakdown. GAG determinations were carried outwith the dimethylmethylene blue assay (Farndale, et al., Biochim.Biophys. Acta. 883: 173-177 (1986)).

Synovia were dissected from the knee joints, fixed in 70% ethanol,dehydrated, embedded in paraffin, sectioned at 5 μm and stained withhematoxylin and eosin.

An average of 2.5 ng human IRAP per knee was measured in joint lavages 4days following transplant of MFG-IRAP/HIG 82 cells. Contralateral,control knees receiving naive HIG-82 cells had no detectable human IRAP(FIG. 11). To determine whether the observed level of IRAP expressionwas sufficient to inhibit the effects of IL-1 in vivo, increasingconcentrations of IL-1β (0-100 pg) were injected into both the controland IRAP knees. As is shown in FIG. 12 a, injection of hrIL-1β intocontrol knees provoked a marked leukocytosis which was stronglysuppressed in the genetically modified knees. There was also astatistically significant reduction in the white blood cell count inknees containing MFG-IRAP/HIG 82 cells which had not been injected withIL-1. This may reflect the influence of IRAP upon the slightinflammatory effect of injecting cells into joints. The degree ofsuppression by IRAP decreased as the amount of injected hrIL-1βincreased, in keeping with the competitive mode of inhibition existingbetween IRAP and IL-1. No dose-response for hrIL-1β alone is evident inthese particular experiments because this specific batch of IL-1 wasespecially effective in eliciting maximal response even at the lowestdose used.

Examination of cytospins (FIGS. 12 b, 12 c) revealed that most of theinfiltrating leukocytes were neutrophils and monocytes. Thesepreparations also serve to illustrate the efficiency with whichleukocytosis was suppressed by the IRAP gene. Ten times the volume oflavage fluid is represented on the cytospin obtained from theIRAP-producing knees (FIG. 12 c) compared to the non-IRAP knee (FIG. 12b).

To determine if intra-articularly expressed IRAP was able to blockcartilage breakdown, the concentration of glycosaminoglycans (GAG) injoint lavages was determined. GAG analyses of the washings from thecontrol and IRAP expressing knees (FIG. 13) confirmed that transfer ofthe IRAP gene partially inhibited breakdown of the cartilaginous matrixin response to IL-1. Again, inhibition was strongest at the lowestconcentrations of IL-1 and was abolished at the highest dose of IL-1(FIG. 13).

In response to 10 pg of injected hrIL-1β, control synovia becamehypertrophic (FIG. 14 a) and hypercellular (FIG. 14 c). The increasedcellularity of the synovia appeared to involve both increased numbers ofsynoviocytes and infiltration by leukocytes. In knees where MFG-IRAP/HIG82 cells were present, these changes were completely suppressed and thesynovia were nearly indistinguishable from control synovia (FIGS. 14 b,14 d).

The ex vivo transfer of the human IRAP gene to the synovial lining ofrabbit knees clearly protects these joints from the pathophysiologicalsequelae of subsequent intra-articular challenge by hrIL-1β.

Measurements of the amounts of IL-1 present in human, recombinantsynovial fluids provide values in the range of 0-500 pg/ml (Westacott,et al., 1990, Ann Rheum Dis. 49: 676-681; Malvak, et al., 1993,Arthritis Rheum 36: 781-789). Thus the amounts of IRAP expressedintra-articularly during the present, short-term experiments should besufficient to block the biological activities of IL-1 at theconcentrations present in human arthritic joints.

Example XIII

This example shows that the level of intraarticular IRAP expressedsubsequent to ex vivo transplantation of synoviocytes transduced withMFG-IRAP is sufficient to inhibit several pathophysiological changesassociated with antigen-induced arthritis of the rabbit knee.Intraarticularly expressed IRAP has both a chondroprotective andanti-inflammatory effect during the acute phase of this disease. Datadisclosed in Example XII support the contention that the invention asdisclosed and claimed is a marked improvement for treating connectivetissue disorders such as arthritis in comparison to delivery of proteinsto the afflicted joint. Example XII shows that ex vivo transfer ofMFG-IRAP to the rabbit knee as disclosed throughout this specificationresults in the intraarticular accumulation of nanogram quantities ofglycosylated, biologically active IRAP. This present example shows thatthis same gene therapy based product inhibits joint pathologies in arabbit model of human rheumatoid arthritis.

Young adult rabbits were subjected to a surgical, partial synovectomy ofthe left knee joint to provide autologous cells. These autologous cellswere used to produce cultures of rabbit synovial fibroblasts (type Bsynoviocytes) from these biopsies as described in Example V and ExampleIX. Subconfluent cultures were then transduced by infection withMFG-IRAP. Expression of the transene was confirmed by measuring theconcentrations of human IRAP in the conditioned media; values typicallyrange from 100-500 ng IRAP/10⁶ cells/3 days. Sister cultures ofsynoviocytes from the same animal were infected with a BAG virusencoding the lac Z and neo^(r) marker genes, and then selected forgrowth in the presence of G418 (1 mg/ml) to serve as controls.Untransduced synoviocytes were also used as additional controls.

During the period that the cells were being grown and transduced, thedonor rabbits were sensitized to ovalbumin by a series of twointradermal injections of 5 mg ovalbumin emulsified in adjuvant, giventwo weeks apart. Two weeks after the second injection, an acutemonarticular arthritis was initiated by the injection of 5 mg ovalbumindissolved in 1 ml saline into the right knee joints. By this time theleft, donor knees had regenerated their synovia, and were each injectedwith 1 ml saline as controls.

One day after the onset of arthritis, 10⁷ autologous cells, transducedwith either the IRAP gene, or lac Z and neo genes, were injected intoeach arthritic knee, and each contralateral, non-arthritic knee. Inother control experiments, knees were injected with untransduced,autologous cells. Groups of rabbits were killed 3 and 7 days later,corresponding to the middle and end of the acute phase of thisarthropathy. Knees were lavaged with 1 ml of saline, prior to theremoval of synovial tissue and articular cartilage for analysis.

Intraarticular expression of the MFG-IRAP transgene was evaluated byELISA measurements of human IRAP in the lavage fluids. IRAPconcentrations in the control, non-arthritic knees is shown in FIG. 15.IRAP concentrations in the arthritic knees were always several-foldhigher than in normal knees at both time points (FIG. 15). In bothnon-arthritic and arthritic knees transduced with MFG-IRAP, there was aslight decrease in IRAP expression with time. No human IRAP could bedetected in sera obtained from normal or arthritic rabbits.

During the course of these experiments, the intraarticular concentrationof rabbit IL-1 in arthritic knees was in the range of 100-200 pg/knee(FIG. 16). No IL-1α could be detected by RIA of the lavage fluids. Thusthe concentration of IRAP within these knees exceeded the concentrationof IL-1 by factors of approximately 10-50. Concentrations of IL-1 werelower in day 7 arthritic knees receiving the IRAP gene (FIG. 16),suggesting that IRAP had inhibited an autocrine amplification loop.

Two major pathologies predominate in the rheumatoid joint: loss ofarticular cartilage and inflammation. The former occurs through acombination of reduced synthesis and synergistic degradation of thecartilaginous matrix. Whereas inflammation is manifest as a synovitisaccompanied by influx of leukocytes into the joint space.

The onset of antigen-induced arthritis in this Example was accompaniedby cartilage destruction, as reflected in the increasedglycosaminoglycan (GAG) content of the lavage fluids (FIG. 17 a), andreduced synthesis of cartilage proteoglycans, as reflected by loweruptake of ³⁵SO₄ ²⁻ (FIG. 17 b). Knees expressing the MFG-IRAP transgene,but not control knees, were substantially protected from these changes.GAG release (FIG. 17 a) was inhibited 55% on day 4 and 32% on day 7.Suppression of GAG synthesis (FIG. 17 b) was inhibited by 68% on day 4and 100% on day 7. The MFG-IRAP transgene also strongly reduced theinflux of leukocytes into the joint space (FIG. 18), an effect that wasstronger at day 4 (65% inhibition) than at day 7 (38% inhibition);indeed, the difference at day 7 failed to reach statisticalsignificance.

The MFG-IRAP construct is utilized to exemplify the presently claimedinvention. In addition to this construct, the ex vivo based teachings ofthis specification have been utilized to transfer to synovial cells andexpress in vivo DNA sequences encoding human IL-1α, human TNF-α solublereceptor Types I and II, vIL-10, growth hormone, IL-6, Lac Z andneo^(r).

Example XIV

The methods disclosed throughout this specification were utilized toexpress MFG-human IL-1 soluble receptor type I and type II constructs(with neo^(r)) within in vitro cultured synoviocytes. These transfectedsynoviocytes produce 1-2 ng/10⁶ cells of IL-1 soluble receptor types Iand II, following neo-selection. The additional methods disclosedthroughout this specification may be utilized to procure in vivoexpression data regarding these MFG-human IL-1 soluble receptor type Iand type II constructs.

Example XV

Rabbits were injected intraarticularly in one knee joint with a specificviral or non-viral vector disclosed in Table 2. Contralateral knees wereinjected with a control, usually with the identical viral or non-viralvector with a different passenger gene. At intervals from 2 days to 2weeks following intraarticular injection, rabbits were sacrificed andthe knee joints harvested and stained with X-Gal to assay for LacZexpression. The results are depicted in Table 2. The recombinantadenovirus vector comprising a CMV-LacZ fusion and the recombinant HSVvector comprising a CMV-LacZ fusion generated the highest expressionlevel subsequent to intraarticular injection. The recombinant retroviralvector, MFG-LacZ, was not expressed in vivo, lending credence to theconcept that retroviral vectors require actively dividing cells duringthe infection process and the concomitant low mitotic activity ofsynoviocytes in the joint lining.

However, an intra-articular injection of MFG-IRAP to synovial cells ofan inflamed joint space supported retroviral transduction. Injection ofMFG-IRAP into an inflamed rabbit knee lead to the intraarticularaccumulation of about 0.5 ng/knee at 7 days post injection. Thecontralateral knee did not express human IRAP. The example shows a MoMLVbased retrovirus can be used for in vivo gene delivery to inflammedjoints.

TABLE 2 EXPRESSION In Vitro In Vivo LAC Z In Vivo DURATION VECTORPROMOTER cells (%) LEVEL (Days) Retrovirus (MFG) LTR 20-30 0 0 HSV CMV 1(toxic) +++ 5-7 Adenovirus CMV 100 +++ ≧14 Liposome CMV 20-30 + 1-2(DC-chol) None CMV 0 ± 1-2 (naked DNA) Level of in vivo expression wasevaluated subjectively on a scale of 0-+++, based upon the degree ofstaining with X-Gal. LTR = viral long terminal repeat CMV =cytomegalovirus

Example XVI

Vector Construction and Virus Production

To facilitate efficient secretion of human interleukin-lβ(hIL-lβ), a DNAfragment encoding mature hIL-Lβ, amino acids 117-269 of the unprocessedprotein was linked to sequences encoding the prepro leader peptide(amino acids 1-31) of human parathyroid hormone (hPTH). To remove thepotential for N-linked glycosylation, amino acids 7 through 9 of maturehIL-1β were changed from the glycosylation consensus sequence,Asn-Cys-Ser, to Gln-Ala-Ser. The preprolL-1β coding region was PCRamplified and inserted into the Nco I and BamH I restriction sites ofthe MEG retroviral vector according to the methods of Dranoff, Proc.Nat. Acad. Sci. U.S.A. 90: 3539-3543 (1993) and Robbins et al., Annalsof the New York Academy of Sciences 716: 72-89 (1993). The upstreamprimer (gccaccATGgTACCTGCA; SEQ ID NO:7) contained nucleotides 1-12 ofthe 5′ end of the hPTH leader sequence (shown in caps.), with the fourthresidue changed from an A to a G and an additional 6 nucleotides toaccommodate the recognition sequence for Neo I (underlined). Thedownstream primer (AGCACAGGATCCTCTGGGTAC; SEQ ID NO:8) corresponded tosequences in the pCDNAI vector adjacent to the 3′ end of the hIL-1βcoding region which were modified to contain a BamH I recognitionsequence (underlined). To allow positive selection of retrovirallytransduced cells,. a DNA fragment containing an internal ribosome entrysite, as described by Ghattas et al., Mol Cell. Biol. 11: 5848-5849(1991). (IRES) 5′ to the cDNA encoding neomycin phosphotransferase (neó)was inserted into the BamH I site of the MFG-hIL-1β plasmid, immediatelydownstream of the hIL-1β coding region. The resulting plasmid construct(pDFG-hIL-1β-neo) allows for expression of both the hIL-1β and neo^(r)gene products from a single polycistronic transcript initiated from theupstream retroviral long terminal repeat as shown by Robbins et al.,715: 72-89 (1993), Tahara et al., J. Immunol. 154: 6466-6477 (1994) andZitvogel et al., Hum Gene Ther. 5:1493-1506 (1994).

A 293-based retroviral packaging cell line, BING, was utilized forproduction of recombinant amphotropic retrovirus, as described by Pearet al., Proc. Natl. Acad. Sci. U.S.A. 90: 8392-8396 (1993).

Transduction of Synoviocytes

Synovial cells were grown to ˜75% confluence in a 25 cm² flaskcontaining 4 ml of Ham's F12 medium with 10% FCS and 1% P/S. The cellswere retrovirally infected as previously described by Bandara et al.,Proc. Natl. Acad. Sci. U.S.A. 90: 10764-10768 (1993). Infected cellswere then selected in medium containing G418 at 0.5 mg/ml.

Synovial Cell Harvest and Transplantation

Partial surgical synovectomies were performed on anesthetized rabbits bysharp dissection via the medial parapatellar approach described by Kanget al., Methods in Molecular Biology: Gene Therapy Protocols, Totowa,N.J., Paul Robbins editor, pp. 357-368 (1996). The harvested tissue wasdigested in 0.2% clostridial collagenase for 2 hrs at 37° C., and thesynoviocytes pelleted by centrifugation. The recovered cells wereresuspended and cultured in Ham's F12 medium with 10% FCS and 1% P/S. Inother experiments, an established line of rabbit synovial fibroblasts,HIG-82, was used. HIG-82 is described in Georgescu et al., In vitro 24:1015-1022 (1988).

For intraarticular transplantation, cells were treated with trypsin,washed, and resuspended in Gey's balanced salt solution (GBSS) to afinal concentration of between 10⁶ to 10⁷ cells per ml. A 1 ml sample ofcell suspension was injected through the patellar tendon into the kneejoints of recipient rabbits.

Biological Analyses

To lavage rabbit knee joints, 1 ml of GBSS plus 10 mM EDTA was injectedinto the joint space through the patellar tendon. After manipulation ofthe joint, the needle was reinserted and the fluid aspirated. Leukocytesin recovered lavage fluids were counted using a hemocytometer. The whiteblood cell types were analyzed by light microscopy of Diff-Quick-stained(Baxter Scientific Products) cytospins. Human IL-1β concentrations inconditioned media, lavage fluids and blood sera were measured asdirected using ELISA kits from R & D Systems. Rabbit IL-1β levels weredetermined using an RIA kit (Cytokine Sciences Inc.).

To measure rabbit TNFα, 96-well plates were coated overnight at 4° C.with goat anti-rabbit TNFα polyclonal antibody (Pharmingen) at aconcentration of 8 ug/ml in 0.1 M NaHCO₃, pH 8.2. The plates were thenwashed twice with PBS containing 0.05% Tween® 20, and then blocked byincubating overnight at 4° C. with PBS plus 10% FCS. After washing twicewith PBS/Tween® 20 solution, standards and samples were added andincubated overnight at 4° C. The plates were then washed four times withPBS/Tween® 20. Detection of the captured antigen was facilitated by theaddition of biotinylated goat anti-rabbit TNFα polyclonal antibody(Pharmingen) at 4 ug/mi in PBS with 10% FCS and incubation 45 min atroom temperature. Plates were then washed six times followed by theaddition of 100 μl/well of avidin-peroxidase (Intergen) diluted 1:400 inPBS/Tween® 20, for 30 min at room temperature. After washing 8 timeswith PBS/Tween® 20, 100 μl/well of TMBlue substrate solution (Intergen)was added and incubated for 20 min at room temperature. Colordevelopment was stopped by the addition of 50 μl/well 1N H₂SO₄, andplates were read at 450 nm.

To quantitate glycosaminoglycans (GAGs) released into the joint space asa result of cartilage proteoglycan degradation, lavage fluids were firstdigested overnight with papain at 60° C. and hyaluronidase for 2 hrs at37° C. GAG concentrations were measured by the 1,9 dimethylmethyleneblue method of Farndale et al., Biochim. Biophys. Acta. 883: 173-177(1986).

To measure proteoglycan synthesis rates, articular cartilage was firstshaved from the femoral condyles and weighed. Approximately 30 mg ofcartilage was then incubated in 500 μl of Neuman Tytell serumless mediumwith 40 μCi of ³⁵SO₄ ⁻² for 24 hrs at 37° C. Afterward, the media wererecovered and stored at −20° C. Proteoglycans were extracted from thecartilage shavings by incubation for 24 hrs in 0.5 ml of 0.5 M NaOH at4° C. with gentle shaking. Following chromatographic separation ofunincorporated ³⁵SO₄ ⁻² using PD-10 columns (Pharmacia), radiolabeledGAGs released into the culture media or recovered by alkaline extractionwere quantitated using scintillation counting.

For histological analyses, tissues harvested from dissected knees werefirst fixed in 10% formalin for 24 hrs. Tissues containing bone andcartilage were subsequently decalcified by incubation in EDTA. The fixedtissues were imbedded in paraffin, sectioned at 5 μm and stained witheither hematoxylin and eosin or toluidine blue.

Immunohistochemical Staining

To identify transduced synoviocytes following transplantation, cultureswere fluorescently labeled by incubation for 5 min in 0.1% PKH2 (Sigma)in GBSS. Following inactivation of the PKH2 by mixing with 10 mls 100%FCS, the labeled cells were washed three times in Ham's F12 medium with10% FCS and resuspended in 1 ml of GBSS for subsequent intraarticularinjection. This procedure resulted in detectable fluorescence in about70-80% of the synoviocytes. To identify cells in the synovium expressinghIL-1β, synovial tissue was first harvested and fixed in 2%paraformaldehyde for 1 hr followed by 30% sucrose for 12 hrs, and frozensectioned at 5 μm. The sections were then blocked with normal horse 1 gin BSA (0.5% bovine serum albumin, 0.15% glycine in phosphate bufferedsaline [PBS]) for 45 min, rinsed 3× with BSA and incubated for 1 hr withpolyclonal goat IgG anti human IL-1β (R & D Systems) in BSA. Thesections were rinsed 3× with BSA and incubated with biotinylated donkeyanti-goat 1 g for 1 hr in BSA. The sections were then incubated withstreptavadin Cy3.18 (Amersham) in BSA, washed 3× with BSA, 3× with PBSand analyzed by fluorescence microscopy.

Ex Vivo hIL-1β Gene Transfer Using HIG-82 Cells

To permit rapid evaluation of the effects of constitutively elevatedexpression of hIL-1β in the rabbit knee joint, the lapine synovial cellline, HIG-82, was initially used for ex vivo gene delivery. Theallogeneic HIG-82 line has been shown previously to only give short termtransgene expression following transplant. (See, for example, Bandara etal., Proc. Nat'l Acad. Sci. USA 90: 10764-10768 (1993)). For theseexperiments, HIG-82 cells were infected in vitro with the retroviralvector DFG-hIL-1-neo which contains the cDNAs for hIL-1β and neomycinphosphotransferase (neo^(r)). Transduced cells were then positivelyselected by culture in media containing G418. ELISA measurements ofmedia conditioned by resistant cultures showed the production of greaterthan 150 ng hIL-1β per 10⁶ cells per 48 hours. Approximately 10⁷ of theHIG-82, hIL-1β+, neo+ cells were then transplanted into one knee jointof nine NZW rabbits by a single intraarticular injection through thepatellar tendon. As a negative control, 10⁷ HIG-82 cells transduced withthe BAG retrovirus, which carries the coding regions for β-galactosidase(lacZ) and neo′, were injected into the contralateral knee joints ofeach rabbit. The methods of Price, et al. describe how to make and usethe BAG virus. “Lineage Analysis in the vertebrate nervous system byretrovirus-mediated gene transfer” Proc. Nat'l. Acad. Sci. U.S.A.,84:156-160 (1987). To monitor the intraarticular accumulation of hIL-1βin the synovial fluids as well as other physiological parameters ofarthritis, knee joints were lavaged at 3 and 7 days posttransplantation. The animals were sacrificed at 7 days post-transplantto permit internal examination of the knee joints.

Relative to the lacZ+ controls, knees receiving hIL-1β+ cells becameswollen within 24 hrs and developed a severe inflammatory arthritiswhich persisted until the animals were sacrificed. ELISA measurements oflavage fluids recovered from each of the rabbits demonstrated meanlevels of ˜20 pg/ml of hIL-β in knees receiving IL-1β+ cells at both 3and 7 days post transplantation (FIG. 19 a), while no measurable hIL-1βwas observed in any control knees. Consistent with elevated levels ofIL-1β, leukocytic infiltration into the joint space was increased by 20and 60 fold on days 3 and 7, respectively (FIG. 19 b), with mean whiteblood cell (WBC) counts for the hIL-1β+ knees exceeding 1.0×10⁸ per mlof lavage fluid on both days. Following dissection of the knee joints atday 7, all the hIL-1β+ knees were found to contain extensive nodularsynovial hypertrophy accompanied by purulent synovial fluid, while eachof the control knees appeared normal. Over expression of hIL-1β was alsonoted to have a significant impact on cartilage matrix proteoglycans,with a greater than 7 fold increase in glycosaminoglycans (GAGs)released into synovial fluids in hIL-1β knees relative to controls(assigned a value of 1) at both day 3 and day 7 (FIG. 19 c).Additionally, proteoglycan synthesis in articular cartilage inhIL-1β+knees was inhibited by nearly 60% relative to controls (FIG. 19d).

Relative GAG synthesis by articular cartilage shavings recovered fromknees following dissection at 7 days post-transplant are shown in FIG.19 d. Synthesis rates for hIL-1β+ knee are expressed relative to thecontralateral lacZ controls which were assigned a value of 100. Allvalues are expressed as the mean±S.E.M.

Histological examination of the synovial and subsynovial tissues fromhIL-1β+knees showed that the synovium was substantially thickened, withvillous projections at the surface. The hypertrophied synovium wasfibrous and contained an expanded population of synovial fibroblasts anda profuse infiltration of neutrophils. Scattered lymphocytes were alsoobserved near the subsynovial layer. In many of the rabbits, attachmentof the expanded synovium to the femoral cartilage and bone was seen,suggestive of early pannus formation.

Inflammation, polymorphonuclear leukocytosis, synovial hypertrophy andhyperplasia, as well as proteoglycan degradation and synthesisinhibition are effects that have been previously attributed to thepresence of elevated IL-1β levels. The occurrence of thesepathophysiological responses only in joints receiving IL-1β+ clearlyindicates that the hIL-1β coding region was successfully transferred tothe rabbit knees and expressed in a biologically active form.

Ex Vivo hIL-1β Gene Transfer Using Autologous Synoviocytes

To facilitate longer intraarticular expression of hIL-1β, autologoussynoviocytes rather than HIG-82 cells were used for gene delivery.Synovium was surgically harvested from the right knee joints of 15rabbits, and the synoviocytes of each rabbit were cultured individually.A portion of the synoviocytes from each donor was then infected with theDFG-hIL-1β-neo retrovirus and selected in media containing G418. Thelevel of hIL-1β from the infected cell cultures from each rabbit rangedfrom 100 to 200 ng per 10⁶ cells per 48 hrs.

In preliminary experiments, 10⁷ hIL-1β+synovial cells were autograftedinto the left knees of the respective donor rabbits, and as control asimilar number of autologous naive cells were injected into the rightknees. Of three rabbits initially tested, however, one died after 6 daysand a second had to be sacrificed for heath concerns after 12 days. Allof the rabbits had diarrhea, were febrile, listless, and had undergoneconsiderable weight loss. In addition to a severe arthritic condition inthe knees receiving hIL-1β+cells, postmortem examination showed thepresence of intestinal wall thinning, suggestive of toxic shock.Therefore, for subsequent experiments only 2.5×10⁶ transduced or naivesynoviocytes were introduced into test and control knees. Groups ofrabbits were sacrificed at 7, 14 and 28 days post-transplantation andanalyzed for hIL-1β expression and its physiological effects on thejoint.

Gene Expression In Vivo Using Autologous Cells

Autologous transplantation of transduced synoviocytes resulted insubstantially higher levels of intraarticular hIL-1β expression thanthose achieved by the use of allogeneic HIG-82 cells. As shown in FIG.20, ELISA measurements detected a mean level of approximately 100 pg ofhIL-1β per ml of lavage fluid in animals sacrificed at both 7 and 14days post-transplant. Between week 2 and 4 however, hIL-1β expressionappears to have been abruptly lost, since no measurable hIL-β was foundin knee lavages of any of the rabbits sacrificed at day 28. Rabbit IL-1β(rIL-1β) production was observed to parallel closely that of hIL-1βtransgene expression. Lavage fluids recovered from knees receivinghIL-1β+cells contained over 200 pg/ml of rIL-1β at both day 7 and 14. Byday 28, however, rabbit IL-1β had returned to background levels. Nodetectable human or rabbit IL-1β was measured in any of the controlknees or in blood sera.

To observe the expression of the hIL-1β transgene in vivo, transducedsynoviocyte cultures were fluorescently labeled in vitro with PKDH 2 andautografted into rabbit knee joints; following sacrifice at three daypost-transplant, synovial sections were immunohistochemically stainedfor hIL-1β. As shown in FIGS. 21 a-c the regions of the synovium whichstain positively for hIL-1β correspond closely with those containingfluorescent cells, demonstrating that the transduced cells have indeedengrafted into the synovial lining and continue to express hIL-1β.Regions which stain for hIL-1β without fluorescence likely representtransplanted synoviocytes unlabeled by the PKH2 in vitro. Morespecifically FIG. 21 a shows fluorescent microscopy of sectionedsynovium dissected from a rabbit knee at 3 days post-transplant of PKH2labeled hIL-1β+, neo+ synoviocytes. Labeled engrafted cells fluorescegreen. FIG. 21 b shows microscopy of the same section shown in FIG. 21 afollowing immunohistochemical staining for hIL-1β. Regions stainingpositive for hIL-1β fluoresce red. FIG. 21 c presents an overlay of theimages of FIG. 21 a and 21 b; this shows that regions staining positivefor hIL-1β closely correspond with regions of PKH2 labeled cells.

Local and Systemic Effects of hIL-1β Expression

Within 24 hrs of transplantation, all knees receiving hIL-1β+cellsbecame noticeably swollen and by 48 hrs displayed a greater than 30%increase in size relative to contralateral control knees that persistedfor approximately 18 to 20 days (FIG. 21 d). Internally, the pathologyof the knee joints front rabbits sacrificed at 7 and 14 days was akin tothat seen with the HIG-82 cells, but noticeably worse (FIG. 21 e). Thesynovial fluid was thick and suppurative, and the synovium was massivelythickened and lobular. In the 14 day rabbits, numerous attachments couldbe seen by gross inspection to penetrate the cartilage and bone on thesides of the femoral condyles. Furthermore, in nearly all of the 7 and14 day rabbits the anterior compartment of the lower leg was filled witha cream colored, caseinous exudate. The muscle tissue surrounding theexudate was often bleached of color, suggestive of local necrosis.Histological examination showed that the substance war comprisedprimarily of densely packed neutrophils within a loose fibrinous matrix.From further dissection of the leg and knee joint, it appeared that theexudate was an accumulation; of the leukocytic infiltrate from the jointspace which penetrated the anterior compartment following partialrupture of the joint capsule.

Although all the animals receiving the hIL-1β+ cells developed a severearthritis and were symptomatically identical during the first two weeksfollowing transplantation, these changes resolved rapidly following theloss of hIL-1β expression at between days 14 and 28. In three of thefive rabbits sacrificed on day 28 the joint capsules appeared relativelynormal by gross examination. The remaining two rabbits also showedimprovement, but still exhibited synovial hypertrophy and had remnantsof an exudate in the anterior compartment.

The nature of the white blood cell infiltration into the joint space ofthe hIL-1β+ knees varied considerably among the groups of rabbits. Anextraordinarily high number of infiltrating leukocytes were observed inrecovered knee washings from rabbits sacrificed within the first twoweeks (FIG. 22 a), with mean levels of 2.6×10⁸ per ml on day 7 and5.2×10⁸ per ml on day 14, an approximately 400 and 2600 fold increase,respectively, over control knees. Leukocytes in synovial fluids at thesetime points were predominantly polymorphonuclear. By day 28, theleukocytosis in the joint space of the hIL-1+ knees had abatedconsiderably, but still remained ˜10 fold higher than controls and wascomprised primarily of mononuclear cells.

Analysis of the effect of hIL-1β over expression on cartilageproteoglycans showed that during the approximately two week period ofexpression of the hIL-β transgene mean levels of GAGs released into thesynovial fluids were greater than 15 fold higher in hIL-1β+ knees overcontrols (assigned a value of 1) (FIG. 22 b). By day 28, GAG release inthe IL-1 knees had returned to that of the controls. Similarly, GAGsynthesis rates were sharply inhibited in the first two weeks, reaching80% repression at day 14 as based on the relative GAG synthesis levelsmeasured from articular cartilage shavings (FIG. 22 c). Synthesis ratesfor hIL-1 knees are expressed relative to contralateral controls whichwere assigned a value of 100. By day 28 significant recovery of thesynthesis was observed.

Consistent with the presence of a systems, inflammatory response, meanelevated erythrocyte sedimentation rates were increased greater than 25fold at both 7 and 14 days (FIG. 22), but by day 21 had returned to nearnormal levels. The rabbits also developed fevers which peaked on orbefore day 7 and gradually subsided by day 28 (FIG. 22 e). Additionally,the rabbits experienced a mean 15% loss in body weight by day 12relative to day 0 (FIG. 22 f) which increased slightly thereafter.

Histological Analysis

FIG. 23 a illustrates normal synovium, against which others arecompared, at 7 days post transplantation of naive synoviocytes.

Histological examination revealed a number of discrete changes in thesynovium and the composition of the inflammatory cells infiltrating thetissue of the joint capsule during the four weeks followingtransplantation of the hIL-1β+ cells. Similar to the one weekHIG-82-IL-1β knees, the synovium from autografted rabbits sacrificed atone week was fibrous and severely hypertrophied, expanded by synovialcell hyperplasia and large numbers of infiltrating neutrophils (FIG. 23b). Scattered lymphocytes and other mononuclear cells were alsoobserved. In rabbits sacrificed at week two, the thickened synoviumshowed signs of neo-vascularization and lobular thickening of thesynovium. A large population of infiltrating neutrophils was stillpresent, but appeared somewhat concentrated toward the outer edges ofthe expanded synovium. Toward the subsynovium, diffuse populations oflymphocytes were observed along with the formation of lymphoid foci(FIG. 23 c and d; FIG. 23 d shows a higher magnification of FIG. 23 c,showing lymphoid (bottom) and neutrophilic infiltration (top), andsynovial hypertrophy and hyperplasia). A highly aggressive pannus wasalso seen in all the rabbits, which had attached to and eroded allperiarticular bone surfaces on the femoral condyles as well as regionsof cartilage. Typically, the most invasive pannus formation occurredlaterally and medially near the edge of the articulating surface. Atthese regions, severe degradation of cartilage could be seen as well aspenetration of the pannus through the bone and into the marrow (FIG. 23g and h). FIG. 23(g) shows a section of femoral condyle from knee at 14days post-transplant of naive synoviocytes. Cartilage (left) was shavedfor GAG synthesis assays. FIG. 23(h) shows a section of femoral condylefrom hIL-1β+ knee at 14 days post-transplant. Highly aggressive pannusis shown eroding cartilage and subchondral bone. Although articularcartilage showed substantial loss of metachromasia followingtoluidine-blue staining, cartilage without attached pannus formationshowed little apparent structural damage (data not shown).

Considerable histological variability was observed among the 28 dayrabbits. In the two rabbits, which by gross examination appearedarthritic, the synovial tissue in general remained somewhat thickenedand vascularized, and contained both peri- and subsynovial lymphoidfollicles (FIG. 23 e). The infiltrating neutrophil population, however,had diminished significantly. Within the subsynovium, concentrations ofmacrophages were observed whose cytoplasm was greatly enlarged andappeared to contain numerous lipid-filled vesicles, perhaps suggestiveof tissue remodeling. A thin lining of normal synovial cells appeared tohave reformed over much of the thickened synovial mass. In a fewisolated pockets, the synovial layer contained high local concentrationsof neutrophils, suggesting that a few of the transplanted synoviocytescontinued to express hIL-1β. Pannus invasion and bone erosion were stillevident in cross sections of the femoral condyles of these rabbits.Although cartilage sections from the tibial plateau and femoral condylesshowed a loss of metachromasia following toluidine-blue staining,regions without attached pannus appeared, at least structurally, intactand unaffected.

In the remaining three 28 day rabbits, a clear reduction in the severityof synovitis was evident (FIG. 23 f). A normal lining of synovial cellshad reformed over the synovial layer. The synovial layer itself wasstill somewhat thickened and fibrous; however, the expanded synovialcell and infiltrating neutrophil and macrophage populations werenoticeably absent. Lymphoid follicles were still present, but theirnumber and size were significantly reduced. Bone and cartilage erosionswere still present, as well as a moderate loss of toluidine-bluestaining in articular cartilage.

This example demonstrates that sustained, interarticular, synovialexpression of IL-1β can produce all the major pathologies associatedwith human RA. Intraarticularly manifestations included intenseinflammation, breakdown of the cartilaginous matrix, inhibition ofmatrix synthesis, leukocytosis of the joint space and synovium, synovialhyperplasia, hypertrophy, neo-vascularization and fibrosis, and theformation of lymphoid follicles and highly aggressive pannus formationwith erosion of the articular cartilage and periarticular bone.Extraarticular and systemic pathologies include periarticular loss ofbone, elevated temperature and elevated erythrocyte sedimentation rate,loss of weight, diarrhea and fever.

From this data, it is clear that the production of hIL-1β from thetransgene stimulated local synthesis and secretion of rabbit IL-1β in aparacrine fashion, making the total IL-1β content of the affected jointsbetween 300 to 400 pg/ml. This compares to values of 0.5 to 5 ng/mlreported for human rheumatoid synovial fluid Hopkins, et al. “Cytokinesin Synovial Fluid I. The Presence of Biologically Active andImmunoreactive IL-1, Clin. Exp. Immunol. 72:422 (1988). Furthermore, theIL-1 induced rTNFα, levels exceeded 600 pg/ml, compared to the 0 to 7ng/ml range reported in RA by Saxne, et al. “Detection of Tumor NecrosisFactor a but not Tumor Necrosis Factor b in Rheumatoid ArthritisSynovial Fluid and Serum,” Arthritis Rheum. 31:1041 (1988). Thus, theintraarticular concentration of IL-1 and TNFα in human RA and our modelappear roughly equivalent. These findings indicate that this hIL-1β genetransfer model not only reflects the potential for IL-1β to contributeto RA pathogenesis, but also provides a close approximation of the RAcytokine milieu and, thus, a novel vehicle to assess the efficacy of RAtherapeutics.

Since TNFα has been shown to be a potent stimulator of IL-1 and GM-CSFproduction in cultured RA synovial fibroblasts, it has been put forththat TNFα is at the top of the inflammatory cytokine cascade thatmediates the pathology of RA (See, for example, Brennan, et al.“Inhibitory effects of TNFα antibodies on synovial cell interleukin-1production in rheumatoid arthritis” Lancet 2:244 (1989); Haworth, et al.“Expression of granulocyte-macrophage colony-stimulating factor inrheumatoid arthritis: regulation by tumor necrosis factor-α,” Eur. J.Immunol.21:2575 (1991); and Maini, et al. “Beneficial effects of tumornecrosis factor-alpha (TNF-α) blockade in rheumatoid arthritis (RA),”Clin. Exp. Immunol. 101:207 (1995). The results of experiments describedhere, however, demonstrate that elevated intraarticular levels of IL-1can stimulate both IL-1 and TNFα production and that upon removal ofIL-1, inflammatory sequelae lessen in severity even in the presence ofelevated TNFα. This shows that in the hierarchy of inflammatorycytokines, IL-1 is most likely the principal mediator of RA-likepathology in the rabbit knee.

The very marked systemic changes, which in the early experimentsincluded serious illness and death of one animal, were quite striking.Intravenous injection of IL-1β is known to induce both fever andcachexia, suggesting that IL-1β was not totally contained within thejoint capsule of the rabbits. However, extraarticular human or rabbitIL-1β or rTNFα was not detected in the sera of any of the animals.Therefore, these effects most likely arise either from the chronicpresence of very low levels (<3.0 pg/ml) of these cytokines in thebloodstream or elevated levels of another effector molecule such asIL-6. IL-1 is a potent inducer of IL-6, which is the most abundantcytokine in human rheumatoid synovial fluids. IL-6 is an importantmediator of the acute phase response, which accounts for the elevatedESR, and has also been implicated as an inducer of fever and cachexia insome animal systems Hirano, et al. “Biological and clinical aspects ofinterleukin-6,” Immunol Today 11:443 (1990).

The results presented in this example clearly illustrate the capacity ofIL-1β to serve as an effector molecule in RA and suggest that agentswhich block the interarticular activity of IL-1β will be therapeutic.These results also serve to validate the use of gene transfer as a meansto study the in vivo action of gene products associated with thepathogenesis of arthritis, and the rabbit model of the presentinvention.

Example XVII

The procedures of Example III were repeated substituting cDNA encodingthe following genes instead of IRAP: sIL-IR Type I; sIL-IR Type II;sTNF-αR Type II; vIL-10; TIMP-2; CTLA4; FasL; and iNOS. Briefly, an MFGretroviral vector was constructed containing cDNA encoding the gene ofinterest selected from the list above. The MFG constructs were then usedto infect synovial cells recovered from rabbit knees. Various methodswere used to determine whether the gene expression occurred in thecells. Immunohistochemistry, bioassay and western analysis were used insome cases; a determination was made as to whether the gene wasexpressed. ELISA and NO production were used in some cases; aquantitative determination of the level of gene expression was made.Results are presented in Table 3 below.

TABLE 3 Gene Expression in Synovial Cells Following Retroviral GeneTransfer Method of Presence/amount of Gene detection product of interestsTNF-alpha receptor type I ELISA 50 ng per 10⁶ cells sTNF-alpha receptortype II ELISA 30 ng per 10⁶ cells sIL-1 receptor type I ELISA 5 ng per10⁶ cells sIL-1 receptor type II ELISA 5 ng per 10⁶ cells vIL-l0 ELISA50 ng per 10⁶ cells TIMP-2 Bioassay Positive CTLA4-Ig Western PositiveFasL Immunohisto. Positive iNOS Western Positive iNOS NO production 20μm per 10⁶ cells

Example XVIII

The animal model procedures of Examples XI and XVI were repeated usingIL-6 and TNF-α.

FIG. 24 a shows the expression of murine IL-6 (mIL-6) in knees injectedwith mIL-6 versus control knees. Several ng of mIL-6 were found in theinjected knees, while mIL-6 in control knees was below the limit ofdetection. Thus, mIL-6 was being expressed in the injected knees. FIG.24 b shows that the amount of GAG released was much greater in the mIL-6knee after 3 days, but was equal to the control knees after 7 days.

FIG. 25 a shows the leukocyte infiltration of knees injected with murineTNF-α (mTNF-α) versus control knees. The leukocyte infiltration in themTNF-α knees was about four to five times that of control knees afterboth 3 and 7 days, indicating that the TNF-α was being expressed in abiologically active form and causing inflammation. FIG. 25 b showsinhibition of cartilage proteoglycan synthesis in the knees receivingthe mTNF-α.

These data demonstrate that the gene therapy methods disclosed andclaimed in the present invention can be used to modulate the diseaseprocess in an animal model of arthritis. In turn, these Examples enablethe claimed gene therapy based treatment of connective tissuepathologies and systemic indices of inflammation within the afflictedjoint(s). It will be appreciated by those skilled in the art that thisinvention provides a method of introducing into a target cell of amammalian host in vitro, or in the alternative in vivo, at least onegene which codes for proteins and/or RNA with therapeutic properties.This method includes employing genes having DNA that is capable ofmaintenance and expression.

It will be appreciated by those skilled in the art that this inventionprovides a method of introducing at least one gene encoding a productinto at least one target cell of a mammalian host for treating aconnective tissue condition of the mammalian host.

It will be understood by those skilled in the art that this inventionprovides a method to repair and regenerate the connective tissue of amammalian host.

It will be further understood that the present invention discloses exvivo and in vivo techniques for delivery of a DNA sequence of interestto the target cells of the mammalian host. The ex vivo techniqueinvolves prior removal and culture of target autologous cells, in vitroinfection of the DNA sequence, DNA vector or other delivery vehicle ofinterest into the target cells, followed by transplantation to themodified target cells to the target joint of the mammalian host, so asto effect in vivo expression of the gene product of interest. The invivo technique bypasses the requirement for in vitro culture of targetcells, instead relying on direct transplantation of the DNA sequence,DNA vector or other delivery vehicle to the target in vivo cells, thuseffecting expression of the gene product of interest.

It will be further understood that this invention provides a method toproduce an animal model for the study of connective tissue pathology.

It will be appreciated by those persons skilled in the art that thisinvention provides a method of using and a method of preparing numerousgenes including a gene encoding soluble interleukin-1 receptor that iscapable of binding to and neutralizing substantially all isoforms ofinterleukin-1, and thus substantially protect cartilage of a mammalianhost from pathological degradation. In addition, it will be understoodby those persons skilled in the art that the method of using the gene ofthis invention will reduce inflammation, protect soft tissues of thejoint and suppress the loss of bone that occurs in patients sufferingwith arthritis.

It will be appreciated by those persons skilled in the art that theviral vectors employed in the present invention may be employed totransfect synovial cells in vivo or in culture, such as by directintraarticular injection or transplantation of autologous synovial cellsfrom the patient transduced with one or more vectors carrying one ormore genes.

It will also be understood that a class of DNA sequences, as describedthroughout this specification, including but not limited to IRAP, mayuse the claimed methods to effect reduction of inflammation, protectsoft tissues of the joint and suppress the loss of bone that occurs inpatients suffering with arthritis.

The present invention provides a method of preparing various vectors,both viral and non viral, that contain DNA sequences encoding fornumerous genes of interest. These genes are known by those skilled inthe art to be useful in the therapeutic treatment of various connectivetissue disorders. The present invention demonstrates that infection oftarget cells with these vectors results in expression of the genes invivo when target cells are returned to the host. Alleviation of symptomscommon to numerous connective tissue disorders is then observed. Thus,the methods of the present invention provide a method of treating a hostthrough the use of target cells transfected with a vector coding forvarious therapeutic genes.

The present invention also provides a method for establishing an animalmodel for the study of connective tissue disorders. This model alsoprovides for the preparation of viral or non-viral vectors herecontaining DNA sequences encoding for numerous genes known to causesymptoms representative of those associated with connective tissuedisorders. Injection of these vectors into an animal result in theexpression of the gene and the onset of the symptoms. Injection can beaccomplished by in vivo means, by directly injecting the vector into thehost, or by ex vivo means, by transfecting target cells, such assynoviocytes with the vectors and injecting the transfected cells intothe host.

Whereas particular embodiments of this invention have been describedabove for purposes of illustration, it will be evident to those personsskilled in the art that numerous variations of the details of thepresent invention may be made without departing from the invention asdefined in the appended claims.

1. A method for treating arthritis in a mammalian host, comprising:generating a recombinant viral vector comprising a DNA sequence encodingsoluble IL-1 receptor operatively linked to a promoter; infecting invitro population of autologous synovial cells with said recombinantviral vector resulting in a population of transduced synovial cells; andtransplanting said transduced synovial cells by intraarticular injectionto an arthritic joint space of a mammalian host, such that expression ofsaid DNA sequence in said joint space results in reduction of cartilagedestruction or reduction in synovitis.
 2. The method of claim 1, whereinsaid soluble IL-1 receptor is selected from the group consisting ofsoluble IL-1 receptor Type 1 and soluble IL-1 receptor Type II.
 3. Themethod of claim 1, wherein said recombinant viral vector is anadenovirus.
 4. The method of claim 1, wherein transplantation of thetransduced cells is by intraarticular injection.
 5. The method of claim1, further including the step of storing said population of transducedcells prior to transplantation.
 6. The method of claim 5, wherein saidpopulation of transduced cells are stored in 10% DMSO under liquidnitrogen prior to transplantation.
 7. The method according to claim 1,wherein the viral vector is a retroviral vector.
 8. The method accordingto claim 1, wherein the viral vector is an adeno-associated viralvector.
 9. The method according to claim 1, wherein the viral vector isa herpes simplex viral vector.
 10. A method for treating-arthritis in amammalian host, comprising: generating a recombinant plasmid vectorcomprising a DNA sequence encoding soluble IL-1 receptor operativelylinked to a promoter; transforming in vitro population of synovial cellswith said recombinant plasmid vector resulting in a population oftransformed synovial cells; and transplanting said transformed synovialcells by intraarticular injection to an arthritic joint space of amammalian host, such that expression of said DNA sequence in said jointspace results in reduction of cartilage destruction or reduction insynovitis.
 11. The method of claim 10, further including the step ofstoring said population of transformed cells prior to transplantation.12. The method of claim 11, wherein said population of transformed cellsare stored in 10% DMSO under liquid nitrogen prior to transplantation.